Patent application title: IDENTIFICATION OF PDE3 MODULATOR RESPONSIVE CANCERS
Inventors:
Xiaoyun Wu (Cambridge, MA, US)
Heidi Greulich (Cambridge, MA, US)
Assignees:
THE BROAD INSTITUTE, INC.
IPC8 Class: AC12Q16886FI
USPC Class:
1 1
Class name:
Publication date: 2021-12-02
Patent application number: 20210371935
Abstract:
The present disclosure features methods for identifying pateints having a
hyperproliferative disease, disorder, or condition responsive to
phosphodiesterase 3 (PDE3) and schlafen family member 12 (SLFN12) complex
formation. The hyperproliferative disease, disorder, or condition may be
cancer in a patient including glioblastoma, melanoma, ovarian cancer,
cervical cancer, sarcoma, or hematopoietic cancers, such as acute myeloid
leukemia. Those responsive diseases, disorders, or conditions may be
identified using the biomarker AIP and/or TRRAP in combination with those
biomarkers pertinent to phosphodieseterase 3 and schlafen family member
12 complexes which may be formed by PDE3 modulation with certain active
compounds. Expression of combinations of these biomarkers have been shown
to correlate with active compound (e.g., PDE3 modulator, PDE3A modulator,
PDE3B modulator) sensitivity.Claims:
1. A method of identifying a subject having a hyperproliferative disease,
disorder, or condition responsive to PDE3A-SLFN12 complex formation or
PDE3B-SLFN12 complex formation are provided comprising obtaining one or
more cells of the hyperproliferative disease, disorder, or condition from
the subject and detecting: (i) the expression of aryl hydrocarbon
receptor interacting protein (AIP) polypeptides or polynucleotides and/or
transformation/transciption domain associated protein (TRRAP)
polypeptides or polynucleotides in the cell; (ii) the expression of
phosphodiesterase 3A (PDE3A) polypeptides or polynucleotides or the
expression of phosphodiesterase 3B (PDE3B) polypeptides or
polynucleotides in the cell relative to a reference, and (iii) the
expression of Schlafen family member 12 (SLFN12) polypeptides or
polynucleotides in the cell relative to the reference; wherein said
hyperproliferative disease, disorder, or condition is characterized as
responsive to said complex formation if: (i) AIP and/or TRRAP are
expressed in the cell, (ii) the expression of PDE3A and/or PDE3B is
increased relative to the reference, and (iii) the expression of SLFN12
is increased relative to the reference.
2. The method according to claim 1, wherein said method further comprises detecting: (ii) the expression of PDE3A polypeptides or polynucleotides in the cells relative to a reference, and the disease, disorder, or condition characterized as responsive to is selected as responsive to PDE3A-SLFN12 complex formation if: (i) AIP and/or TRRAP are expressed in the cell, (ii) the expression of PDE3A is increased relative to the reference, and (iii) the expression of SLFN12 is increased relative to the reference.
3. The method according to claim 1, wherein said method comprises detecting: (ii) the expression of PDE3B polypeptides or polynucleotides, and the disease, disorder, or condition characterized as responsive to is selected as responsive to PDE3B-SLFN12 complex formation if: (i) AIP and/or TRRAP are expressed in the cell, (ii) the expression of PDE3B is increased relative to the reference, and (iii) the expression of SLFN12 is increased relative to the reference.
4. The method according to claim 1, comprising the detection of AIP and TRRAP expression in the cell, wherein the hyperproliferative disease, disorder, or condition is characterized as responsive to said complex formation if: (i) AIP and TRRAP are expressed, (ii) the expression of PDE3A and/or PDE3B is increased relative to the reference, and (iii) the expression of SLFN12 is increased relative to the reference.
5. A method of killing or reducing the survival of a cell selected as responsive to PDE3A-SLFN12 complex formation or PDE3B-SLFN12 complex formation comprising contacting said cell with a PDE3 modulator, wherein said cell is selected as responsive to said PDE3 modulator when said cell: (i) expresses AIP and/or TRRAP polypeptides or polynucleotides, (ii) has increased expression of PDE3A and/or PDE3B polypeptides or polynucleotides relative to a reference, and (iii) has increased expression of SLFN12 polypeptides or polynucleotides relative to the reference.
6. The method according to claim 5, wherein said cell is selected as responsive to said PDE3 modulator when said cell: (i) expresses AIP and TRRAP polypeptides or polynucleotides, (ii) has increased expression of PDE3A and/or PDE3B polypeptides or polynucleotides relative to a reference, and (iii) has increased expression of SLFN12 polypeptides or polynucleotides relative to the reference.
7. The method according to claim 5, wherein said cell is selected as responsive to said PDE3 modulator when said cell: (i) expresses AIP and TRRAP polypeptides or polynucleotides, (ii) has increased expression of PDE3A polypeptides or polynucleotides relative to a reference, and (iii) has increased expression of SLFN12 polypeptides or polynucleotides relative to the reference.
8. The method according to claim 5, wherein said cell is selected as responsive to said PDE3 modulator when said cell: (i) expresses AIP and TRRAP polypeptides or polynucleotides, (ii) has increased expression of PDE3B polypeptides or polynucleotides relative to a reference, and (iii) has increased expression of SLFN12 polypeptides or polynucleotides relative to the reference.
9. A method for the treatment or prevention of a hyperproliferative disease, disorder, or condition in a subject comprising administering to said subject a PDE3 modulator, wherein said subject is identified as having a hyperproliferative disease, disorder, or condition that is responsive to the PDE3 modulator with the method according to claim 1.
10. The method of claim 1, wherein the PDE3 modulator is 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-on- e (DNMDP) or (6S)-5-[4'-fluoro-2-(trifluoromethyl)biphenyl-4-yl]-6-methyl-3,6-dihydro-- 2H-1,3,4-oxadiazin-2-one (Compound X).
11-13. (canceled)
14. The method of claim 1, wherein said hyperproliferative disease, disorder, or condition is a myeloproliferative disease.
15. The method of claim 1, wherein said cell is a cancer cell.
16. The method of claim 15, wherein said cancer cell is a melanoma-, endometrium-, lung-, hematopoetic-/lymphoid-, ovarian-, cervical-, soft-tissue- sarcoma-, urinary tract, pancreas-, thyroid-, kidney-, glioma-, glioblastoma-, or breast-cancer cell.
17. The method of claim 15, wherein said cancer cell is a melanoma-, glioma-, glioblastoma-, ovarian-, sarcoma-, acute myeloid leukemia-, or lung adenocarcinoma cell.
18. The method of claim 1, wherein said PDE3 modulator is administered orally or by intravenous injection.
19. (canceled)
20. The method of claim 1, wherein said cancer cell is collected from a tissue sample, a blood sample, or a plasma sample.
21. A kit for identifying a subject having cancer as responsive to PDE3A-SLFN12 complex formation or PDE3B-SLFN12 complex formation, the kit comprising a first capture reagent that binds AIP polypeptide and/or a second capture reagent that binds TRRAP polypeptide.
22. The kit according to claim 21, wherein said kit comprises a third capture reagent that binds PDE3A polypeptide and/or a fourth capture reagent that binds PDE3B polypeptide and a fifth capture reagent that binds SLFN12 polypeptide.
23. The kit according to claim 21, wherein said kit comprises a third capture reagent that binds PDE3A polypeptide or a fourth capture reagent that binds PDE3B polypeptide and a fifth capture reagent that binds SLFN12 polypeptide.
24. The kit according to claim 21, wherein said kit further comprises a third capture reagent that binds SLFN12 polypeptide.
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of priority under 35 U.S.C. .sctn. 119 to U.S. App. No. 62/901,090, filed on Sep. 16, 2019 and U.S. App. No. 62/754,290, filed Nov. 1, 2018, each of which is hereby incorporated by reference in its entirety.
FIELD OF DISCLOSURE
[0002] The present disclosure relates to the identification of cells (e.g., cancer cells) responsive to complex formation between certain phosphodiesterase 3 proteins (e.g., PDE3A, PDE3B) and schlafen family member 12 (SLFN12) proteins by identifying cells that express certain biomarkers implicated in complex formation. Specifically, cells identified as expressing the aryl hydrocarbon receptor interacting protein (AIP) and/or transformation/transcription domain associated protein (TRRAP) are implicated in the complex formation which results in apoptosis. Methods of treatment or prevention of hyperproliferative diseases, disorders, or conditions associated with these cells are also provided comprising administration of certain chemical agents (e.g., PDE3A modulators) to those cells identified as responsive to complex formation.
BACKGROUND
[0003] Cancer kills over 550,000 people in the United States and over 8 million people world-wide each year. New agents, including small molecules, molecules that impact tissue-specific growth requirements, and immunomodulatory agents, have been shown to benefit a subset of patients whose cancers have unique genomic mutations or other characteristics. Unfortunately, many cancer patients are still left without effective therapeutic options.
[0004] One approach to identify new anti-cancer agents is phenotypic screening to discover novel small molecules displaying strong selectivity between cancer cell lines, followed by predictive chemogenomics to identify the cell features associated with drug response. In the 1990s, Weinstein and colleagues demonstrated that the cytotoxic profile of a compound can be used to identify cellular characteristics, such as gene-expression profiles and DNA copy number, that correlate with drug sensitivity. The ability to identify the features of cancer cell lines that mediate their response to small molecules has strongly increased in recent years with automated high-throughput chemosensitivity testing of large panels of cell lines coupled with comprehensive genomic and phenotypic characterization of the cell lines. Phenotypic observations of small-molecule sensitivity can be linked to expression patterns or somatic alterations.
[0005] Despite advances in targeted therapies and immunotherapies, certain cancers, such as metastatic melanoma, remain deadly diseases. For example, metastatic melanoma has a 5-year survival rate of only 20%. New therapeutic modalities are therefore needed. These new modalities may be based on new mechanisms of cancer cell killing. For example, some phosphodiesterase 3A (PDE3A) modulators may cause complex formation between PDE3A peptide and schlafen family member 12 (SLFN12) or similar complex formation between PDE3B peptide and schlafen family member 12 (SLFN12) in cancer cells. This complex formation may result in induction of apoptosis. However, inhibition of PDE3 enzymatic activity alone is insufficient to cause cancer cell killing, as neither PDE3 knockout nor treatment with most previously-characterized PDE3A inhibitors kills cancer cells. Thus, in contrast to traditional targeted therapies that leverage cancer cell dependencies created by genomic alteration, these PDE3 modulation therapies cause induced apoptosis via PDE3A-SLFN12 or PDE3B-SLFN12 ("PDE3A/B-SLFN12") complex formation. Moreover, such apoptotic induction does not occur in all cancer cells expressing PDE3A and SLFN12 indicating an incomplete understanding of the mechanistic underpinnings of this cell death.
[0006] Methods of characterizing malignancies at a molecular level are useful for stratifying patients, thereby quickly directing them to effective therapies. Improved methods for predicting the responsiveness of subjects having cancer are urgently required.
SUMMARY
[0007] In accordance with the foregoing objectives and others, the present disclosure provides methods of identifying cells of a hyperproliferative disease, disorder, or condition, such as cancer cells responsive to PDE3A-SLFN12 complex formation or PDE3B-SLFN12 complex formation, methods for the treatment or prophylaxis of hyperproliferative diseases, disorders, or conditions identified as being responsive to complex formation, and kits for the determination and treatment of hyperproliferative diseases, disorders, or conditions identified as being responsive to complex formation. Without wishing to be bound by theory, it is believed that cells lacking the aryl hydrocarbon receptor interacting protein (AIP) and/or the transformation/transciption domain associated protein (TRRAP) have decreased or no sensitivity to complex formation or have decreased or no complex formation following contact of the cell with an active compound that typically induces such formation. Certain PDE3 modulatory compounds (e.g., PDE3A modulators, PDE3B modulators, DNMDP, compounds disclosed in WO2019/025562, which is hereby incorporated by reference in its entirety and particularly in relation to compounds of general formula (1), may be able to induce complex formation between PDE3A and SLFN12 or PDE3B and SLFN12 in cancer cells when those cells express AIP and/or TRRAP, which may result apoptosis of the cancer cells.
[0008] Apoptosis may be induced in cells expressing the AIP polypeptide or polynucleotide particularly in relation to cells expressing schlafen family 12 (SLFN12) and phosphodiesterase 3A (PDE3A) or cells expressing schlafen family member 12 (SLFN12) and phosphodiesterase 3B (PDE3B) since AIP is implicated in PDE3A-SLFN12 or PDE3B-SLFN12 complex formation. It has also been found that apoptosis may be induced in cells expressing transformation/transciption domain associated protein (TRRAP) polypeptide or polynucleotide in relation to cells expressing schlafen family member 12 (SLFN12) and phosphodiesterase 3A (PDE3A) or cells expressing schlafen family member 12 (SLFN12) and phosphodiesterase 3B (PDE3B) since TRRAP is implicated in formation of or response to complex formation as well.
[0009] Methods of identifying a subject having a hyperproliferative disease, disorder, or condition, such as a cancer responsive to PDE3A-SLFN12 complex formation or PDE3B-SLFN12 complex formation, are provided comprising detecting:
[0010] (i) the expression of aryl hydrocarbon receptor interacting protein (AlP) polypeptides or polynucleotides and/or transformation/transciption domain associated protein (TRRAP) polypeptides or polynucleotides;
[0011] (ii) the expression of phosphodiesterase 3A (PDE3A) polypeptides or polynucleotides or the expression of phosphodiesterase 3B (PDE3B) polypeptides or polynucleotides in the cells relative to a reference, and
[0012] (iii) the expression of schlafen family member 12 (SLFN12) polypeptides or polynucleotides in the cells relative to a reference;
wherein the hyperproliferative disease, disorder, or condition is characterized as responsive to the complex formation complex formation if:
[0013] (i) AIP and/or TRRAP are expressed,
[0014] (ii) the expression of PDE3A and/or PDE3B is increased relative to the reference, and
[0015] (iii) the expression of SLFN12 is increased relative to the reference.
In certain implementations, the method may comprise obtaining one or more cells (e.g., cancer cells) of the hyperproliferative disease, disorder, or condition from the subject and detecting:
[0016] (i) the expression of aryl hydrocarbon receptor interacting protein (AlP) polypeptides or polynucleotides and/or transformation/transciption domain associated protein (TRRAP) polypeptides or polynucleotides;
[0017] (ii) the expression of phosphodiesterase 3A (PDE3A) polypeptides or polynucleotides or the expression of phosphodiesterase 3B (PDE3B) polypeptides or polynucleotides in the cells relative to a reference, and
[0018] (iii) the expression of schlafen family member 12 (SLFN12) polypeptides or polynucleotides in the cells relative to a reference;
wherein the hyperproliferative disease, disorder, or condition is characterized as responsive to the complex formation complex formation if:
[0019] (i) AIP and/or TRRAP are expressed,
[0020] (ii) the expression of PDE3A and/or PDE3B is increased relative to the reference, and
[0021] (iii) the expression of SLFN12 is increased relative to the reference.
In some embodiments, the hyperproliferative disease, disorder, or condition is characterized as responsive to complex formation if both AIP and TRRAP are expressed in the cells. In certain implementations, expression of AIP and/or TRRAP may be determined by comparison of expression to the reference and the hyperprofliferative disease, disorder, or condition is characterized as responsive to complex formation if:
[0022] (i) there is no loss of AIP and/or TRRAP expression relative to the reference (e.g., the expression levels of AIP and/or TRRAP in the cell is more than 50% of the expression level in the reference or more than 90% of the expression levels in the reference or more than 100% of the expression levels in the reference),
[0023] (ii) the expression of PDE3A is increased relative to the reference, and
[0024] (iii) the expression of SLFN12 is increased relative to the reference. In some embodiments, the method of identifying a subject having a hyperproliferative disease, disorder, or condition, such as cancer responsive to PDE3A-SLFN12 complex formation, may comprise obtaining one or more cells (e.g., cancer cells) of the hyperproliferative disease, disorder, or condition from the subject and detecting:
[0025] (i) the expression of aryl hydrocarbon receptor interacting protein (AlP) polypeptides or polynucleotides and/or transformation/transciption domain associated protein (TRRAP) polypeptides or polynucleotides,
[0026] (ii) the expression of phosphodiesterase 3A (PDE3A) polypeptides or polynucleotides relative to a reference, and
[0027] (iii) the expression of schlafen family member 12 (SLFN12) polypeptides or polynucleotides relative to a reference;
wherein the hyperproliferative disease, disorder, or condition is characterized as responsive to said complex formation if:
[0028] (i) AIP and/or TRRAP are expressed,
[0029] (ii) the expression of PDE3A is increased relative to the reference, and
[0030] (iii) the expression of SLFN12 is increased relative to the reference. In some embodiments, the hyperproliferative disease, disorder, or condition is characterized as responsive to complex formation if both AIP and TRRAP are expressed.
[0031] In various implementations, the method of identifying a subject having a hyperproliferative disease, disorder, or condition, such as cancer responsive to PDE3B-SLFN12 complex formation, may comprise obtaining one or more cells (e.g., cancer cells) of the hyperproliferative disease, disorder, or condition from the subject and detecting:
[0032] (i) the expression of aryl hydrocarbon receptor interacting protein (AlP) polypeptides or polynucleotides and/or transformation/transciption domain associated protein (TRRAP) polypeptides or polynucleotides,
[0033] (ii) the expression of phosphodiesterase 3B (PDE3B) polypeptides or polynucleotides relative to a reference, and
[0034] (iii) the expression of schlafen family member 12 (SLFN12) polypeptides or polynucleotides relative to a reference;
wherein the hyperproliferative disease, disorder, or condition is characterized as responsive to said complex formation complex formation if:
[0035] (i) AIP and/or TRRAP are expressed,
[0036] (ii) the expression of PDE3B is increased relative to the reference, and
[0037] (iii) the expression of SLFN12 is increased relative to the reference. In some embodiments, the hyperproliferative disease, disorder, or condition is characterized as responsive to chemically induced complex formation if both AIP and TRRAP are expressed.
[0038] The cells of the subject may be collected from a tissue sample, a blood sample, or a plasma sample.
[0039] Methods of killing or reducing the survival of a cell (e.g., cancer cell) are also provided wherein the cancer cell is selected as responsive to PDE3A/B-SLFN12 complex formation comprising contacting the cancer cell with a PDE3 modulator (e.g., a PDE3A modulator, a PDE3B modulator), wherein the cell is selected as responsive to the PDE3A/B-SLFN12 complex formation when the cell expresses AIP and/or TRRAP polypeptides or polynucleotides, has increased expression of SLNF12 polypeptides or polynucleotides relative to a reference, and has increased expression of PDE3A or PDE3B relative to the reference. Typically, the PDE3 modulator (e.g., PDE3A modulator, PDE3B modulator) may be able to induce PDE3A/B-SLFN12 complex formation leading to apoptosis of the cell following contact. In some embodiments, the cancer cell is selected as responsive to PDE3A-SLFN12 complex formation if the cell has increased expression of PDE3A and increased expression of SLFN12 relative to a reference. In some embodiments, the cell is selected as responsive to PDE3B-SLFN12 complex formation if the cell has increased expression of PDE3B and SLFN12 relative to a reference. Many PDE3A modulators also directly bind PDE3B proteins and a PDE3A modulator may be used to induce complexation between SLFN12 and PDE3B.
[0040] In some embodiments, methods for the treatment or prevention of hyperproliferative disease, disorder, or condition (e.g. cancer) in a subject are provided comprising administering to the subject a PDE3 modulator (e.g., PDE3A modulators, PDE3B modulators), wherein the subject is identified as having a hyperproliferative disease, disorder, or condition that is responsive to the PDE3 modulator by obtaining one or more cells of the hyperproliferative disease, disorder, or condition (e.g. cancer) from the subject (e.g., by obtaining a sample from the subject) and detecting:
[0041] (i) the expression of aryl hydrocarbon receptor interacting protein (AlP) polypeptides or polynucleotides and/or transformation/transciption domain associated protein (TRRAP) polypeptides or polynucleotides,
[0042] (ii) the expression of phosphodiesterase 3B (PDE3B) polypeptides or polynucleotides relative to a reference, and
[0043] (iii) the expression of Schlafen family member 12 (SLFN12) polypeptides or polynucleotides relative to a reference;
wherein the hyperproliferative disease, disorder, or condition is characterized as responsive to said complex formation complex formation if:
[0044] (i) AIP and/or TRRAP are expressed,
[0045] (ii) the expression of PDE3B is increased relative to the reference, and
[0046] (iii) the expression of SLFN12 is increased relative to the reference. In some embodiments, the hyperproliferative disease, disorder, or condition is characterized as responsive to the PDE3 modulator if both AIP and TRRAP are expressed. The PDE3A modulator may comprise, for example, 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin- -3(2H)-one (DNMDP). In certain implementations, expression of AIP and/or TRRAP may be determined by comparison of expression to the reference and the hyperprofliferative disease, disorder, or condition is characterized as responsive to complex formation if:
[0047] (i) there is no loss of AIP and/or TRRAP expression relative to the reference (e.g., the expression levels of AIP and/or TRRAP in the cell is more than 50% of the expression levels in the reference or more than 90% of the expression levels in the reference or more than 100% of the expression levels in the reference),
[0048] (ii) the expression of PDE3A is increased relative to the reference, and
[0049] (iii) the expression of SLFN12 is increased relative to the reference.
[0050] The expression of any biomarker (e.g., AIP, TRRAP, PDE3A, PDE3B, SLFN12) may be detected by a method selected from the group consisting of immunoblotting, mass spectrometry, immunoprecipitation quantitative PCR, Northern Blot, microarray, enzyme-linked immunosorbent assay (ELISA), in situ hybridization, and combinations thereof. In certain implementations, expression of AIP and/or TRRAP may be determined by comparison to the reference. The cancer cell may be considered to express AIP and/or TRRAP if there is no loss in expression as compared to the reference. In certain implementations, expression of AIP and/or TRRAP may be determined by comparison to the reference and the cell is considered to express AIP and/or TRRAP if there is a small difference (e.g., the cancer cell copy number is within 10% of the copy number of the reference, the cancer cell copy number is within 5% of the reference) between expression in the cancer cell and the reference. Genomics may be used to determine expression and relative expression levels. For example, the cell may be considered to not express AIP and/or TRRAP if the number of copies of the biomarker per cellular genome is less than 1 or less than 2.sup.-1 or less than 2.sup.-2 or less than 2.sup.-3 or less than 2.sup.-4 or less than 2.sup.-5. Conversely, the cell may be considered to express AIP and/or TRRAP if the number of copies of the biomarker per cellular genome is greater than 1 or greater than 2.sup.-1 or greater than 2.sup.-2 or greater than 2.sup.-3 or greater than 2.sup.-4 or greater than 2.sup.-5.
[0051] Such methods allow for the treatment and/or prevention of hyperproliferative disease, disorders, or conditions caused by the proliferation of cells responsive to complex formation and, in particular, complex formation induced by PDE3 modulation. In some embodiments, the cell is a cancer cell. For example, the hyperproliferative disease, disorder, or condition may be selected from bladder, brain, breast, cervical, colorectal, endometrial, esophageal, gallbladder, gastric, glioblastoma, kidney, leukemia (e.g., acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia), liver (e.g., hepatocellular carcinoma, intrahepatic cholangiocarcinoma, angiosarcoma, hemangiosarcoma, hepatoblastoma), lung (e.g., non-small cell lung cancer, small cell lung cancer, mesothelioma), melanoma, ovarian, pancreatic, prostate, multiple myeloma, sarcoma (e.g., osteosarcoma, soft-tissue sacrcoma), thyroid, urinary tract, or uterine cancer. In certain implementations the cancer may be a hematopoietic cancer, such as acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute monocytic leukemia, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.
[0052] Various routes of administration are useful for treatment modalities. In some embodiments, the PDE3 modulator (e.g., PDE3A modulator, PDE3B modulator) is administered orally. In other embodiments, the PDE3 modulator is administered by intravenous injection.
[0053] Kits for identifying a subject having cancer as responsive to complex formation including chemically induced complex formation (e.g., cells responsive to PDE3 modulators, cells responsive to PDE3A modulators, cells responsive to PDE3B modulators) are also provided, wherein the kit comprises a first capture reagent that binds AIP polypeptide and/or a second capture reagent that binds TRRAP polypeptide. In some embodiments, the kit comprises a third capture reagent that binds PDE3A polypeptide and/or a fourth capture reagent that binds SLFN12 polypeptide and/or a fifth capture reagent that binds PDE3B. In some embodiments, the kit further comprises a PDE3 modulator (e.g., PDE3A modulator, PDE3B modulator), such as DNMDP or a compound of WO2019/025562. It will be understood that the numeric identifiers for the capture reagents (e.g., first, second, third, fourth, fifth) do not indicate the total quantity of capture reagents in each kit. The PDE3 modulator may be present in a pharmaceutical formulation sufficient to deliver a therapeutically effective amount to a subject in need thereof.
[0054] In one embodiment, a cancer expressing AIP, which is required for SLFN12/PDE3A complex formation, is identified as responsive to treatment with compound X ((6S)-5-[4'-fluoro-2-(trifluoromethyl)biphenyl-4-yl]-6-methyl-3,6-dihydro- -2H-1,3,4-oxadiazin-2-one).
BRIEF DESCRIPTION OF FIGURES
[0055] FIG. 1 illustrates the reads per kilobase of transcript per million mapped reads (RPKM) for the 49 cell lines measured for PDE3A sensitivity and identifies the biomarker positive cancer cell lines in addition to the HeLa cells. A high proportion of biomarker-positive cell lines are from melanoma patients.
[0056] FIG. 2A shows DNMDP 72 h Cell Titer-Glo assay dose response curves for HeLa cells and the 7 melanoma cell lines showing DNMDP sensitivity. As can be seen, C32 and RVH421 are only partially sensitive. FIG. 2B shows DNMDP 72 h Cell Titer-Glo assay dose response curves for several cell lines sensitive to DNMDP treatment. FIG. 2C shows DNMDP 72 h Cell Titer-Glo assay dose response curves for several cell lines partially sensitive or insensitive to DNMDP treatment.
[0057] FIG. 3A shows the survival of cells as compared to cells with CRISPR KO of SLFN12 following at various DNMDP concentrations. A2058 and SKMEL3 are representative melanoma cell lines. Survival is measured with a 72 h Cell Titer-Glo assay and CRISPR was performed with sg4, SLFN12 CRISPR guide RNA #4. FIG. 3B shows the survival of HeLa cells as compared to PDE3A knockout cells (with and without ectopic PDE3B expression).
[0058] FIG. 4 identifies those CRISPR gene targets resulting in decreased sensitivity for DNMDP cancer cell killing in HeLa cells. AIP, SLFN12, and PDE3A knockout cause the greatest increase in cell survival in the presence of 25 nM DNMDP. The results are plotted as log fold change (LFC) of gene CRISPR guide RNA representation among all genes compared to -log p-values, indicating the likelihood of significance. TRRAP also exhibited a significant increase in cell survival in the presence of 25 nM DNMDP.
[0059] FIG. 5 compares the gene copy number and expression of AlP in the measured cell lines and identifies the UACC257 cell line as lacking AIP expression. As shown in FIG. 2A, UACC257 does not have sensitivity to DNMDP.
[0060] FIG. 6A illustrates the results of the 72-hour Cell Titer-Glo assay with independent AIP CRISPR gRNAs (sg) in HeLa cells. FIG. 6B shows the results of the 72-hour Cell Titer-Glo assay with independent AIP CRISPR gRNAs (sg) in A2058 melanoma cells. FIG. 6C is an immunoblot revealing that AIP knockout decreases PDE3A protein levels in DNMDP-sensitive cell lines.
[0061] FIG. 7 is an immunoblot illustrating that AIP knockout prevents DMNDP induced complex formation. PDE3A immunoprecipitates from HeLa cells transiently transfected with V5-tagged SLNF12 and treated with 10 .mu.M DNMDP.
[0062] FIG. 8A is a graph and FIG. 8B is an image of an immunoblot. The two figures show that that AIP is necessary for Compound X induced cancer cell killing and induction of the PDE3A-SLFN12 complex. FIG. 8A shows that cervical cancer cell viability is reduced at increasing concentration of Compound X. This effect is not observed when AIP is knocked out using CRISPR (KOsg2, KOsg3). FIG. 8B is an image of an immunoblot. An anti-PDE3A antibody was used to pull down a PDE3A-SLFN12 complex. Complex formation between PDE3A and FLAG-tagged SLFN12 was induced in the presence of Compound X and in the presence of DNMDP. Complex formation was not observed when AIP was knocked out using Crispr (KO sg2, KO sg3). HeLa cells were treated with 10 .mu.M DNMDP or 10 .mu.M Compound X.
DETAILED DESCRIPTION
Definitions
[0063] Detailed embodiments of the present disclosure are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative of the disclosure that may be embodied in various forms. In addition, each of the examples given in connection with the various embodiments of the disclosure is intended to be illustrative, and not restrictive.
[0064] All terms used herein are intended to have their ordinary meaning in the art unless otherwise provided. All concentrations are in terms of percentage by weight of the specified component relative to the entire weight of the topical composition, unless otherwise defined.
[0065] As used herein, "a" or "an" shall mean one or more. As used herein when used in conjunction with the word "comprising," the words "a" or "an" mean one or more than one. As used herein "another" means at least a second or more.
[0066] As used herein, all ranges of numeric values include the endpoints and all possible values disclosed between the disclosed values. The exact values of all half integral numeric values are also contemplated as specifically disclosed and as limits for all subsets of the disclosed range. For example, a range of from 0.1% to 3% specifically discloses a percentage of 0.1%, 1%, 1.5%, 2.0%, 2.5%, and 3%. Additionally, a range of 0.1 to 3% includes subsets of the original range including from 0.5% to 2.5%, from 1% to 3%, and from 0.1% to 2.5%. It will be understood that the sum of all weight % of individual components will not exceed 100%.
[0067] By "PDE3A polynucleotide" is meant any nucleic acid molecule encoding a PDE3A polypeptide or fragment thereof. An exemplary PDE3A nucleic acid sequence is provided at NCBI Ref: NM_000921.4:
TABLE-US-00001 (SEQ ID NO: 1) 1 gggggccact gggaattcag tgaagagggc accctatacc atggcagtgc ccggcgacgc 61 tgcacgagtc agggacaagc ccgtccacag tggggtgagt caagccccca cggcgggccg 121 ggactgccac catcgtgcgg accccgcatc gccgcgggac tcgggctgcc gtggctgctg 181 gggagacctg gtgctgcagc cgctccggag ctctcggaaa ctttcctccg cgctgtgcgc 241 gggctccctg tcctttctgc tggcgctgct ggtgaggctg gtccgcgggg aggtcggctg 301 tgacctggag cagtgtaagg aggcggcggc ggcggaggag gaggaagcag ccccgggagc 361 agaagggggc gtcttcccgg ggcctcgggg aggtgctccc gggggcggtg cgcggctcag 421 cccctggctg cagccctcgg cgctgctctt cagtctcctg tgtgccttct tctggatggg 481 cttgtacctc ctgcgcgccg gggtgcgcct gcctctggct gtcgcgctgc tggccgcctg 541 ctgcgggggg gaagcgctcg tccagattgg gctgggcgtc ggggaggatc acttactctc 601 actccccgcc gcgggggtgg tgctcagctg cttggccgcc gcgacatggc tggtgctgag 661 gctgaggctg ggcgtcctca tgatcgcctt gactagcgcg gtcaggaccg tgtccctcat 721 ttccttagag aggttcaagg tcgcctggag accttacctg gcgtacctgg ccggcgtgct 781 ggggatcctc ttggccaggt acgtggaaca aatcttgccg cagtccgcgg aggcggctcc 841 aagggagcat ttggggtccc agctgattgc tgggaccaag gaagatatcc cggtgtttaa 901 gaggaggagg cggtccagct ccgtcgtgtc cgccgagatg tccggctgca gcagcaagtc 961 ccatcggagg acctccctgc cctgtatacc gagggaacag ctcatggggc attcagaatg 1021 ggaccacaaa cgagggccaa gaggatcaca gtcttcagga accagtatta ctgtggacat 1081 cgccgtcatg ggcgaggccc acggcctcat taccgacctc ctggcagacc cttctcttcc 1141 accaaacgtg tgcacatcct tgagagccgt gagcaacttg ctcagcacac agctcacctt 1201 ccaggccatt cacaagccca gagtgaatcc cgtcacttcg ctcagtgaaa actatacctg 1261 ttctgactct gaagagagct ctgaaaaaga caagcttgct attccaaagc gcctgagaag 1321 gagtttgcct cctggcttgt tgagacgagt ttcttccact tggaccacca ccacctcggc 1381 cacaggtcta cccaccttgg agcctgcacc agtacggaga gaccgcagca ccagcatcaa 1441 actgcaggaa gcaccttcat ccagtcctga ttcttggaat aatccagtga tgatgaccct 1501 caccaaaagc agatccttta cttcatccta tgctatttct gcagctaacc atgtaaaggc 1561 taaaaagcaa agtcgaccag gtgccctcgc taaaatttca cctctttcat cgccctgctc 1621 ctcacctctc caagggactc ctgccagcag cctggtcagc aaaatttctg cagtgcagtt 1681 tccagaatct gctgacacaa ctgccaaaca aagcctaggt tctcacaggg ccttaactta 1741 cactcagagt gccccagacc tatcccctca aatcctgact ccacctgtta tatgtagcag 1801 ctgtggcaga ccatattccc aagggaatcc tgctgatgag cccctggaga gaagtggggt 1861 agccactcgg acaccaagta gaacagatga cactgctcaa gttacctctg attatgaaac 1921 caataacaac agtgacagca gtgacattgt acagaatgaa gatgaaacag agtgcctgag 1981 agagcctctg aggaaagcat cggcttgcag cacctatgct cctgagacca tgatgtttct 2041 ggacaaacca attcttgctc ccgaacctct tgtcatggat aacctggact caattatgga 2101 gcagctaaat acttggaatt ttccaatttt tgatttagtg gaaaatatag gaagaaaatg 2161 tggccgtatt cttagtcagg tatcttacag actttttgaa gacatgggcc tctttgaagc 2221 ttttaaaatt ccaattaggg aatttatgaa ttattttcat gctttggaga ttggatatag 2281 ggatattcct tatcataaca gaatccatgc cactgatgtt ttacatgctg tttggtatct 2341 tactacacag cctattccag gcctctcaac tgtgattaat gatcatggtt caaccagtga 2401 ttcagattct gacagtggat ttacacatgg acatatggga tatgtattct caaaaacgta 2461 taatgtgaca gatgataaat acggatgtct gtctgggaat atccctgcct tggagttgat 2521 ggcgctgtat gtggctgcag ccatgcacga ttatgatcat ccaggaagga ctaatgcttt 2581 cctggttgca actagtgctc ctcaggcggt gctatataac gatcgttcag ttttggagaa 2641 tcatcacgca gctgctgcat ggaatctttt catgtcccgg ccagagtata acttcttaat 2701 taaccttgac catgtggaat ttaagcattt ccgtttcctt gtcattgaag caattttggc 2761 cactgacctg aagaaacact ttgacttcgt agccaaattt aatggcaagg taaatgatga 2821 tgttggaata gattggacca atgaaaatga tcgtctactg gtttgtcaaa tgtgtataaa 2881 gttggctgat atcaatggtc cagctaaatg taaagaactc catcttcagt ggacagatgg 2941 tattgtcaat gaattttatg aacagggtga tgaagaggcc agccttggat tacccataag 3001 ccccttcatg gatcgttctg ctcctcagct ggccaacctt caggaatcct tcatctctca 3061 cattgtgggg cctctgtgca actcctatga ttcagcagga ctaatgcctg gaaaatgggt 3121 ggaagacagc gatgagtcag gagatactga tgacccagaa gaagaggagg aagaagcacc 3181 agcaccaaat gaagaggaaa cctgtgaaaa taatgaatct ccaaaaaaga agactttcaa 3241 aaggagaaaa atctactgcc aaataactca gcacctctta cagaaccaca agatgtggaa 3301 gaaagtcatt gaagaggagc aacggttggc aggcatagaa aatcaatccc tggaccagac 3361 ccctcagtcg cactcttcag aacagatcca ggctatcaag gaagaagaag aagagaaagg 3421 gaaaccaaga ggcgaggaga taccaaccca aaagccagac cagtgacaat ggatagaatg 3481 ggctgtgttt ccaaacagat tgacttgtca aagactctct tcaagccagc acaacattta 3541 gacacaacac tgtagaaatt tgagatgggc aaatggctat tgcattttgg gattcttcgc 3601 attttgtgtg tatattttta cagtgaggta cattgttaaa aactttttgc tcaaagaagc 3661 tttcacattg caacaccagc ttctaaggat tttttaagga gggaatatat atgtgtgtgt 3721 gtatataagc tcccacatag atacatgtaa aacatattca cacccatgca cgcacacaca 3781 tacacactga aggccacgat tgctggctcc acaatttagt aacatttata ttaagatata 3841 tatatagtgg tcactgtgat ataataaatc ataaaggaaa ccaaatcaca aaggagatgg 3901 tgtggcttag caaggaaaca gtgcaggaaa tgtaggttac caactaagca gcttttgctc 3961 ttagtactga gggatgaaag ttccagagca ttatttgaat tctgatacat cctgccaaca 4021 ctgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgaaaga gagacagaag 4081 ggaatggttt gagagggtgc ttgtgtgcat gtgtgtgcat atgtaaagag atttttgtgg 4141 tttaagtaac tcagaatagc tgtagcaaat gactgaatac atgtgaacaa acagaaggaa 4201 gttcactctg gagtgtcttt gggaggcagc cattccaaat gccctcctcc atttagcttc 4261 aataaagggc cttttgctga tggagggcac tcaagggctg ggtgagaggg ccacgtgttt 4321 ggtattacat tactgctatg caccacttga aggagctcta tcaccagcct caaacccgaa 4381 agactgaggc attttccagt ctacttgcct aatgaatgta taggaactgt ctatgagtat 4441 ggatgtcact caactaagat caaatcacca tttaagggga tggcattctt tatacctaaa 4501 cacctaagag ctgaagtcag gtcttttaat caggttagaa ttctaaatga tgccagagaa 4561 ggcttgggaa attgtacttc agcgtgatag cctgtgtctt cttaatttgc tgcaaaatat 4621 gtggtagaga aagaaaagga aacagaaaaa tcactctggg ttatatagca agagatgaag 4681 gagaatattt caacacaggg tttttgtgtt gacataggaa aagcctgatt cttggcaact 4741 gttgtagttt gtctttcagg ggtgaaggtc ccactgacaa cccctgttgt ggtgttccac 4801 acgctgtttg ttggggtagc ttccatcggc agtctggccc attgtcagtc atgcttcttc 4861 tggccgggga gattatagag agattgtttg aagattgggt tattattgaa agtctttttt 4921 tttgtttgtt ttgttttggt ttgtttgttt atctacactt gtttatgctg tgagccaaac 4981 ctctatttaa aaagttgata ctcactttca atattttatt tcatattatt atatatgtca 5041 tgatagttat cttgatgtaa atatgaagat ttttttgttt ctgtagatag taaactcttt 5101 ttttaaaaaa ggaaaaggga aacattttta taaagttata ttttaatcac catttttata 5161 cattgtagtt ctctccaagc ccagtaagag aatgatgatt catttgcatg gaggtcgatg 5221 gacaaccaat catctacctt ttctaattta aatgataatc tgatatagtt ttattgccag 5281 ttaaatgagg atgctgcaaa gcatgttttt tcactagtaa cttttgctaa ctgaatgaat 5341 tctgggtcca tatctcccag atgaaaaact gttaaccaat accatatttt atagttggtg 5401 tccatttctt tccaacactg tttgttatga ttcttccttg agtacttata tacagacctg 5461 ctcattatct aaacaatctt accttctaag taaaccttga ttgtgatttc cagtttttat 5521 tttctctgac gtagtagaaa ggaatgttta cattaaaaat acttttgttt ctcataaatg 5581 gatattgtac tccccccttt caaagcatta ttttacaata attcatggca ttttaaaaaa 5641 taaggcaaag ataatacgac aaaaaatata catggtttca aggcaaattc tccaataagt 5701 tggaaaatgt aaaaaggatc aagtggatgc agcctctacc taaataatta aaatatattt 5761 cagtatattt ctgaattaac accaggtctt cattatttag aacttactaa attgttttca 5821 ttttcttagt tttacctgtg tatctccatg tttgcaaaaa ttactataag tcaaattttg 5881 ccagtgaatt taactatttt tctttccttg caattaaggg gaaaaaagca tttatcttat 5941 cttctcatac cccttgcatc taagtactta gcaaagtcaa tattttccca ttttccaaat 6001 gcgtccatct ctaacataaa tattaattga acatagagct atgtttggag tgagtggact 6061 ggcaggacag ttggaagtcc atcacagtct attgacagtt tcatcaaagc tgtatagtcc 6121 aactagtggg gcagcttggc tactatggtg gaagtctcag caaactgcct ggttttgttt 6181 gtttgttttg ttttaaggta caggaaataa gaggaataat agtggccaaa gcaattagaa 6241 catcttcatt ccagaactgt gttcagcaat ccaggcagat tgatacattt ttctttaaaa 6301 ataaattgct attacagcta gacgtcaatt gggataaata aagggatgaa gatccactaa 6361 gtttgtgact ttcatacaca cccagtacat ctcaaaggat gctaagggac attttctgcc 6421 agtagagttc tccccctttt tggtgacagc aatattatta tgttcacatc taactccaga 6481 gcttacttcc tgtggtgcca atgtatttgt tgcaatttac tacattttta tatgagccta 6541 tttataggtg ccattaaact caggtctttc aaatgaaaga gtttctagcc cacttaggga 6601 aaaagataat tgtttagaaa accataaaat caatggtagg aaaagttgga actggttacc 6661 tggatgccat ggttctctgt taaataaagt aagagaccag gtgtattctg agtgtcatca 6721 gtgttatttt cagcatgcta ataaatgtct ttccggttat atatctatct aaattaacct 6781 ttaaaatatt ggtttccttg ataaaagcac cacttttgct tttgttagct gtaatatttt 6841 ttgtcattta gataagacct ggtttggctc tcaataaaag atgaagacag tagctctgta 6901 cagggatata tctatattag tcttcatctg atgaatgaag aaattttctc atattatgtt 6961 caagaaagta tttacttcct aaaaatagaa ttcccgattc tgtctatttt ggttgaatac 7021 cagaacaaat ctttccgttg caatcccagt aaaacgaaag aaaaggaata tcttacagac 7081 tgttcatatt agatgtatgt agactgttaa tttgcaattt ccccatattt cctgcctatc 7141 ttacccagat aactttcttt gaaggtaaaa gctgtgcaaa aggcatgaga ctcaggccta 7201 ctctttgttt aaatgatgga aaaatataaa ttattttcta agtaataaaa gtataaaaat 7261 tatcattata aataaagtct aaagtttgaa attattaatt taaaaaaaaa aaaaaaaaa
[0068] By "PDE3A polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at NCBI Ref No. NP_000912.3 that catalyzes the hydrolysis of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). An exemplary human full-length PDE3A amino acid sequence is:
TABLE-US-00002 (SEQ ID NO: 2) MAVPGDAARVRDKPVHSGVSQAPTAGRDCHHRADPASPRDSGCRGCWGDLV LQPLRSSRKLSSALCAGSLSFLLALLVRLVRGEVGCDLEQCKEAAAAEEEE AAPGAEGGVFPGPRGGAPGGGARLSPWLQPSALLFSLLCAFFWMGLYLLRA GVRLPLAVALLAACCGGEALVQIGLGVGEDHLLSLPAAGVVLSCLAAATWL VLRLRLGVLMIALTSAVRTVSLISLERFKVAWRPYLAYLAGVLGILLARYV EQILPQSAEAAPREHLGSQLIAGTKEDIPVFKRRRRSSSVVSAEMSGCSSK SHRRTSLPCIPREQLMGHSEWDHKRGPRGSQSSGTSITVDIAVMGEAHGLI TDLLADPSLPPNVCTSLRAVSNLLSTQLTFQAIHKPRVNPVTSLSENYTCS DSEESSEKDKLAIPKRLRRSLPPGLLRRVSSTWTTTTSATGLPTLEPAPVR RDRSTSIKLQEAPSSSPDSWNNPVMMTLTKSRSFTSSYAISAANHVKAKKQ SRPGALAKISPLSSPCSSPLQGTPASSLVSKISAVQFPESADTTAKQSLGS HRALTYTQSAPDLSPQILTPPVICSSCGRPYSQGNPADEPLERSGVATRTP SRTDDTAQVTSDYETNNNSDSSDIVQNEDETECLREPLRKASACSTYAPET MMFLDKPILAPEPLVMDNLDSIMEQLNTWNFPIFDLVENIGRKCGRILSQV SYRLFEDMGLFEAFKIPIREFMNYFHALEIGYRDIPYHNRIHATDVLHAVW YLTTQPIPGLSTVINDHGSTSDSDSDSGFTHGHMGYVFSKTYNVTDDKYGC LSGNIPALELMALYVAAAMHDYDHPGRTNAFLVATSAPQAVLYNDRSVLEN HHAAAAWNLFMSRPEYNFLINLDHVEFKHFRFLVIEAILATDLKKHFDFVA KFNGKVNDDVGIDWTNENDRLLVCQMCIKLADINGPAKCKELHLQWTDGIV NEFYEQGDEEASLGLPISPFMDRSAPQLANLQESFISHIVGPLCNSYD SA GLMPGKWVEDSDESGDTDDPEEEEEEAPAPNEEETCENNESPKKKTFKRRK IYCQITQHLLQNHKMWKKVIEEEQRLAGIENQSLDQTPQSHSSEQIQAIKE EEEEKGKPRGEEIPTQKPDQ
[0069] Several PDE3A isoforms are known including PDE3A1, PDE3A2, and PDE3A3. PDE3A1 comprises amino acids 146-1141, PDE3A2 isoform 2 comprises amino acids 299-1141, and PDE3A3 comprises amino acids 483-1141 of the full-length PDE3A amino acid sequence. Additionally, spliced transcript variants encoding multiple isoforms have been observed for PDE3A. One such transcript variant has NCBI Ref No. NM_001244683 which has an associated protein sequence (NP_001231612.1):
TABLE-US-00003 (SEQ ID NO: 3) MVTIFSKSWSFYWEKSSGTSITVDIAVMGEAHGLITDLLADPSLPPNVCTS LRAVSNLLSTQLTFQAIHKPRVNPVTSLSENYTCSDSEESSEKDKLAIPKR LRRSLPPGLLRRVSSTWTTTTSATGLPTLEPAPVRRDRSTSIKLQEAPSSS PDSWNNPVMMTLTKSRSFTSSYAISAANHVKAKKQSRPGALAKISPLSSPC SSPLQGTPASSLVSKISAVQFPESADTTAKQSLGSHRALTYTQSAPDLSPQ ILTPPVICSSCGRPYSQGNPADEPLERSGVATRTPSRTDDTAQVTSDYETN NNSDSSDIVQNEDETECLREPLRKASACSTYAPETMMFLDKPILAPEPLVM DNLDSIMEQLNTWNFPIFDLVENIGRKCGRILSQVSYRLFEDMGLFEAFKI PIREFMNYFHALEIGYRDIPYHNRIHATDVLHAVWYLTTQPIPGLSTVIND HGSTSDSDSDSGFTHGHMGYVFSKTYNVTDDKYGCLSGNIPALELMALYVA AAMHDYDHPGRTNAFLVATSAPQAVLYNDRSVLENHHAAAAWNLEMSRPEY NFLINLDHVEFKHERFLVIEAILATDLKKHEDFVAKENGKVNDDVGIDWTN ENDRLLVCQMCIKLADINGPAKCKELHLQWTDGIVNEFYEQGDEEASLGLP ISPFMDRSAPQLANLQESFISHIVGPLCNSYDSAGLMPGKWVEDSDESGDT DDPEEEEEEAPAPNEEETCENNESPKKKTFKRRKIYCQITQHLLQNHKMWK KVIEEEQRLAGIENQSLDQTPQSHSSEQIQAIKEEEEEKGKPRGEEIPTQK PDQ.
In some embodiments, the expression of isoforms of PDE3A in the cell may be measured.
[0070] By "PDE3B polynucleotide" is meant any nucleic acid molecule encoding a PDE3B polypeptide or fragment thereof. An exemplary PDE3B nucleic acid sequence is provided at NCBI Ref: NM_000922.3:
TABLE-US-00004 (SEQ ID NO: 4) 1 gctcgcgcgc ccaacggacc aggctggggc cgtgaggtaa ctgttgcagc cagcggaggt 61 gggaggcgac actgagtctc cagtcccgag aggtgcccga gggaaaagga ggcggcagct 121 aaactggtcc tggagagaag ccccttccgc ccctctcctc agccagcatg tcccggactc 181 cgccgctcct cagtccgcgc ggtggggacc ccgggccgtg gcggccggcg cagccctgac 241 gggttgcgaa ccagggggcg ccccgaacgc gggggttggg gtctgggagc gcgagcggcc 301 gctacggtac gagcggggtg tgctgagtcc cgtggccacc cccggcccca gccatgagga 361 gggacgagcg agacgccaaa gccatgcggt ccctgcagcc gccggatggg gccggctcgc 421 cccccgagag tctgaggaac ggctacgtga agagctgcgt gagccccttg cggcaggacc 481 ctccgcgcgg cttcttcttc cacctctgcc gcttctgcaa cgtggagctg cggccgccgc 541 cggcctctcc ccagcagccg cggcgctgct cccccttctg ccgggcgcgc ctctcgctgg 601 gcgccctggc tgcctttgtc ctcgccctgc tgctgggcgc ggaacccgag agctgggctg 661 ccggggccgc ctggctgcgg acgctgctga gcgtgtgttc gcacagcttg agccccctct 721 tcagcatcgc ctgtgccttc ttcttcctca cctgcttcct cacccggacc aagcggggac 781 ccggcccggg ccggagctgc ggctcctggt ggctgctggc gctgcccgcc tgctgttacc 841 tgggggactt cttggtgtgg cagtggtggt cttggccttg gggggatggc gacgcagggt 901 ccgcggcccc gcacacgccc ccggaggcgg cagcgggcag gttgctgctg gtgctgagct 961 gcgtagggct gctgctgacg ctcgcgcacc cgctgcggct ccggcactgc gttctggtgc 1021 tgctcctggc cagcttcgtc tggtgggtct ccttcaccag cctcgggtcg ctgccctccg 1081 ccctcaggcc gctgctctcc ggcctggtgg ggggcgctgg ctgcctgctg gccctggggt 1141 tggatcactt ctttcaaatc agggaagcgc ctcttcatcc tcgactgtcc agtgccgccg 1201 aagaaaaagt gcctgtgatc cgaccccgga ggaggtccag ctgcgtgtcg ttaggagaaa 1261 ctgcagccag ttactatggc agttgcaaaa tattcaggag accgtcgttg ccttgtattt 1321 ccagagaaca gatgattctt tgggattggg acttaaaaca atggtataag cctcattatc 1381 aaaattctgg aggtggaaat ggagttgatc tttcagtgct aaatgaggct cgcaatatgg 1441 tgtcagatct tctgactgat ccaagccttc caccacaagt catttcctct ctacggagta 1501 ttagtagctt aatgggtgct ttctcaggtt cctgtaggcc aaagattaat cctctcacac 1561 catttcctgg attttacccc tgttctgaaa tagaggaccc agctgagaaa ggggatagaa 1621 aacttaacaa gggactaaat aggaatagtt tgccaactcc acagctgagg agaagctcag 1681 gaacttcagg attgctacct gttgaacagt cttcaaggtg ggatcgtaat aatggcaaaa 1741 gacctcacca agaatttggc atttcaagtc aaggatgcta tctaaatggg ccttttaatt 1801 caaatctact gactatcccg aagcaaaggt catcttctgt atcactgact caccatgtag 1861 gtctcagaag agctggtgtt ttgtccagtc tgagtcctgt gaattcttcc aaccatggac 1921 cagtgtctac tggctctcta actaatcgat cacccataga atttcctgat actgctgatt 1981 ttcttaataa gccaagcgtt atcttgcaga gatctctggg caatgcacct aatactccag 2041 atttttatca gcaacttaga aattctgata gcaatctgtg taacagctgt ggacatcaaa 2101 tgctgaaata tgtttcaaca tctgaatcag atggtacaga ttgctgcagt ggaaaatcag 2161 gtgaagaaga aaacattttc tcgaaagaat cattcaaact tatggaaact caacaagaag 2221 aggaaacaga gaagaaagac agcagaaaat tatttcagga aggtgataag tggctaacag 2281 aagaggcaca gagtgaacag caaacaaata ttgaacagga agtatcactg gacctgattt 2341 tagtagaaga gtatgactca ttaatagaaa agatgagcaa ctggaatttt ccaatttttg 2401 aacttgtaga aaagatggga gagaaatcag gaaggattct cagtcaggtt atgtatacct 2461 tatttcaaga cactggttta ttggaaatat ttaaaattcc cactcaacaa tttatgaact 2521 attttcgtgc attagaaaat ggctatcgag acattcctta tcacaatcgt atacatgcca 2581 cagatgtgct acatgcagtt tggtatctga caacacggcc agttcctggc ttacagcaga 2641 tccacaatgg ttgtggaaca ggaaatgaaa cagattctga tggtagaatt aaccatgggc 2701 gaattgctta tatttcttcg aagagctgct ctaatcctga tgagagttat ggctgcctgt 2761 cttcaaacat tcctgcatta gaattgatgg ctctatacgt ggcagctgcc atgcatgatt 2821 atgatcaccc agggaggaca aatgcatttc tagtggctac aaatgcccct caggcagttt 2881 tatacaatga cagatctgtt ctggaaaatc atcatgctgc gtcagcttgg aatctatatc 2941 tttctcgccc agaatacaac ttccttcttc atcttgatca tgtggaattc aagcgctttc 3001 gttttttagt cattgaagca atccttgcta cggatcttaa aaagcatttt gattttctcg 3061 cagaattcaa tgccaaggca aatgatgtaa atagtaatgg catagaatgg agtaatgaaa 3121 atgatcgcct cttggtatgc caggtgtgca tcaaactggc agatataaat ggcccagcaa 3181 aagttcgaga cttgcatttg aaatggacag aaggcattgt caatgaattt tatgagcagg 3241 gagatgaaga agcaaatctt ggtctgccca tcagtccatt catggatcgt tcttctcctc 3301 aactagcaaa actccaagaa tcttttatca cccacatagt gggtcccctg tgtaactcct 3361 atgatgctgc tggtttgcta ccaggtcagt ggttagaagc agaagaggat aatgatactg 3421 aaagtggtga tgatgaagac ggtgaagaat tagatacaga agatgaagaa atggaaaaca 3481 atctaaatcc aaaaccacca agaaggaaaa gcagacggcg aatattttgt cagctaatgc 3541 accacctcac tgaaaaccac aagatatgga aggaaatcgt agaggaagaa gaaaaatgta 3601 aagctgatgg gaataaactg caggtggaga attcctcctt acctcaagca gatgagattc 3661 aggtaattga agaggcagat gaagaggaat agcgacagtt tgagtaaaag aaaagtcata 3721 ttgaagaagc ccagagggtt gtgcccaggg gcagaaatca ttgcctagtg ttcaccggct 3781 gactctcaac tgaccattcc catgtggaca ggccttaata ctgtgagagg atccttgctc 3841 tgctggcagt ttcccactcc tatgcacttt cacaggaact agaaaactat tcttaaacca 3901 aaaataccat ccgtgttgac ccatgttgca gagcccttac ttaaatcctt cactggtgta 3961 tgaatacttt gtcataatgc tgctttgctg ggtagtgagc tcttattttt cactgggggt 4021 cagctataac taaaaactca agtgacatat ttcagttacc aaagtggcca ggaacttttt 4081 gcttttatga aaatagattc atattgtatt tcccagtgtg tcttttatgt ctttgaatgt 4141 tttggagaaa agtctatgcc tgtctaaaaa tgaatccagt gttgcctttc tgagggattt 4201 ctgctcaatg caatacactg ttcagtgcta ttctcccagc taggtttatc catgaaggac 4261 tgagtgacct ttgttgtatt taacaaaatc caggtgcatc aatttctgat gctttttact 4321 attgtgtatt atctactatg tgtgttttat ttctgctgag agtattcagg tttgccatgg 4381 acatcagaag tttgaattcc agtcttatct tatgttccat ggctgaattt taaagctgtt 4441 taggtttaac aatgaaggga tttattcttt agtcaaaatt gttgttttta ctctagctca 4501 ggattcgtat ttttaaagat ttagttaata tgaacacagc acagatttgt tagaagaaaa 4561 aaaatttgct gtaataccaa aactaacctc atcaaagata cagaaaaaaa gaaatatagt 4621 gagccctaaa ggacacatac attgaataaa taattggaac atgtggttat ctttagatcc 4681 acatcttagc tgtcatttgt tcactctaaa actgatgttc atctttctgt taatttccct 4741 ctgcctaaag actacatgac agaaatgacc tatcactact tattatttct gaagcctaac 4801 tgcaagactg atttctgaga acaagtaaag aactggaata cttatttttc atataaaaat 4861 ctaaatgtgt taataaatca tttcatacaa aagtacatta ttaaataacc acattattaa 4921 aataattgca agaaaatgga ccatatttac aatgttttgt aaacttgcta gtgtgtggat 4981 atgtacccta cttgtgaaat acatttgaag atataaagag cagccaaaat gatggcaaaa 5041 tggtaggcta atattttcta ttattattgg agaacatatc atattttgga atcatgcaat 5101 tttgcacaca gtgaaaccat taattttcca aggtaattcc tttagaatat ggtattggca 5161 tgcagtttct tacttatcta gaatatttgg cttatctgaa agatatcaat ttaagatctc 5221 tggaagtgtt agaatttttg atccttcaca gtgtcaatat ttaatgaatc actaagcttt 5281 atttattaga cgtgttgagt gagtgctgag ttccttgctg ccacttttgt taccattgtc 5341 acacactatg tgtaaaccag tcccaccact tattactaat aaaattttga ctgataattt 5401 atatttgcac ttacaatata tatatcctgt ccttatattt ctctagagta cattttccat 5461 catgtttaag tgtatttctg ctattatttc ctctcctgca gaatacatac aagtgtatgt 5521 gtataaagtc atacatgtac aagcatgcat attgagattg aatcacattt ccatactgtc 5581 tgttatttta ttgggtttta tattgggttt ctttagttta tgttgttttc tcaaaagcag 5641 cattttaaat tacgaatact ggacttattg gatttaatta taaatccaat tactactgga 5701 aactcatttt tacataatat agtccttaaa ttatttaacc cttgctaagt aattgacata 5761 tgtaacaata actagcctaa agaaacccaa aaaagtatct ctcccgagct gaaacttaaa 5821 aattcgtaag tgtaagaaag aatgtgagaa tatattaaat gcacactgta ccattagatg 5881 aaatcttact tgagaaattg ccataagcca tattacagat cttactttgt tactgaatca 5941 gattaatttc ttgttataat aattttcatc ataaattttc tatttttaaa gccgctggta 6001 ctagaaatat tcttttaatg ctatatctat gtacctactg acacattttt ctccataaaa 6061 gtacttttaa aaattacttc atgatttgaa a
[0071] By "PDE3B polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at NCBI Ref No. NP_000913.2. An exemplary human full-length PDE3A amino acid sequence is:
TABLE-US-00005 (SEQ ID NO: 5) MRRDERDAKAMRSLQPPDGAGSPPESLRNGYVKSCVSPLRQDPPRGFFFHL CRFCNVELRPPPASPQQPRRCSPFCRARLSLGALAAFVLALLLGAEPESWA AGAAWLRTLLSVCSHSLSPLFSIACAFFFLTCFLTRTKRGPGPGRSCGSWW LLALPACCYLGDFLVWQWWSWPWGDGDAGSAAPHTPPEAAAGRLLLVLSCV GLLLTLAHPLRLRHCVLVLLLASFVWWVSFTSLGSLPSALRPLLSGLVGGA GCLLALGLDHFFQIREAPLHPRLSSAAEEKVPVIRPRRRSSCVSLGETAAS YYGSCKIFRRPSLPCISREQMILWDWDLKQWYKPHYQNSGGGNGVDLSVLN EARNMVSDLLTDPSLPPQVISSLRSISSLMGAFSGSCRPKINPLTPFPGFY PCSEIEDPAEKGDRKLNKGLNRNSLPTPQLRRSSGTSGLLPVEQSSRWDRN NGKRPHQEFGISSQGCYLNGPFNSNLLTIPKQRSSSVSLTHHVGLRRAGVL SSLSPVNSSNHGPVSTGSLTNRSPIEFPDTADFLNKPSVILQRSLGNAPNT PDFYQQLRNSDSNLCNSCGHQMLKYVSTSESDGTDCCSGKSGEEENIFSKE SFKLMETQQEEETEKKDSRKLFQEGDKWLTEEAQSEQQTNIEQEVSLDLIL VEEYDSLIEKMSNWNFPIFELVEKMGEKSGRILSQVMYTLFQDTGLLEIFK IPTQQFMNYFRALENGYRDIPYHNRIHATDVLHAVWYLTTRPVPGLQQIHN GCGTGNETDSDGRINHGRIAYISSKSCSNPDESYGCLSSNIPALELMALYV AAAMHDYDHPGRTNAFLVATNAPQAVLYNDRSVLENHHAASAWNLYLSRPE YNFLLHLDHVEFKRFRFLVIEAILATDLKKHFDFLAEFNAKANDVNSNGIE WSNENDRLLVCQVCIKLADINGPAKVRDLHLKWTEGIVNEFYEQGDEEANL GLPISPFMDRSSPQLAKLQESFITHIVGPLCNSYDAAGLLPGQWLEAEEDN DTESGDDEDGEELDTEDEEMENNLNPKPPRRKSRRRIFCQLMHHLTENHKI WKEIVEEEEKCKADGNKLQVENSSLPQADEIQVIEEADEEE
[0072] By "SLFN12 polynucleotide" is meant any nucleic acid molecule encoding a SLFN12 polypeptide or fragment thereof. An exemplary SLFN12 nucleic acid sequence is provided at NCBI Ref: NM_018042.4:
TABLE-US-00006 (SEQ ID NO: 6) 1 tttgtaactt cacttcagcc tcccattgat cgctttctgc aaccattcag actgatctcg 61 ggctcctatt tcatttacat tgtgtgcaca ccaagtaacc agtgggaaaa ctttagaggg 121 tacttaaacc ccagaaaatt ctgaaaccgg gctcttgagc cgctatcctc gggcctgctc 181 ccaccctgtg gagtgcactt tcgttttcaa taaatctctg cttttgttgc ttcattcttt 241 ccttgctttg tttgtgtgtt tgtccagttc tttgttcaac acgccaagaa cctggacact 301 cttcactggt aacatatttt ggcaagccaa ccaggagaaa agaatttctg cttggacact 361 gcatagctgc tgggaaaatg aacatcagtg ttgatttgga aacgaattat gccgagttgg 421 ttctagatgt gggaagagtc actcttggag agaacagtag gaaaaaaatg aaggattgta 481 aactgagaaa aaagcagaat gaaagtgtct cacgagctat gtgtgctctg ctcaattctg 541 gagggggagt gatcaaggct gaaattgaga atgaagacta tagttataca aaagatggaa 601 taggactaga tttggaaaat tcttttagta acattctgtt atttgttcct gagtacttag 661 acttcatgca gaatggtaac tactttctga tttttgtgaa gtcatggagc ttgaacacct 721 ctggtctgcg gattaccacc ttgagctcca atttgtacaa aagagatata acatctgcaa 781 aagtcatgaa tgccactgct gcactggagt tcctcaaaga catgaaaaag actagaggga 841 gattgtattt aagaccagaa ttgctggcaa agaggccctg tgttgatata caagaagaaa 901 ataacatgaa ggccttggcc ggggtttttt ttgatagaac agaacttgat cggaaagaaa 961 aattgacctt tactgaatcc acacatgttg aaattaaaaa cttctcgaca gaaaagttgt 1021 tacaacgaat taaagagatt ctccctcaat atgtttctgc atttgcaaat actgatggag 1081 gatatttgtt cattggttta aatgaagata aagaaataat tggctttaaa gcagagatga 1141 gtgacctcga tgacttagaa agagaaatcg aaaagtccat taggaagatg cctgtgcatc 1201 acttctgtat ggagaagaag aagataaatt attcatgcaa attccttgga gtatatgata 1261 aaggaagtct ttgtggatat gtctgtgcac tcagagtgga gcgcttctgc tgtgcagtgt 1321 ttgctaaaga gcctgattcc tggcatgtga aagataaccg tgtgatgcag ttgaccagga 1381 aggaatggat ccagttcatg gtggaggctg aaccaaaatt ttccagttca tatgaagagg 1441 tgatctctca aataaatacg tcattacctg ctccccacag ttggcctctt ttggaatggc 1501 aacggcagag acatcactgt ccagggctat caggaaggat aacgtatact ccagaaaacc 1561 tttgcagaaa actgttctta caacatgaag gacttaagca attaatatgt gaagaaatgg 1621 actctgtcag aaagggctca ctgatcttct ctaggagctg gtctgtggat ctgggcttgc 1681 aagagaacca caaagtcctc tgtgatgctc ttctgatttc ccaggacagt cctccagtcc 1741 tatacacctt ccacatggta caggatgagg agtttaaagg ctattctaca caaactgccc 1801 taaccttaaa gcagaagctg gcaaaaattg gtggttacac taaaaaagtg tgtgtcatga 1861 caaagatctt ctacttgagc cctgaaggca tgacaagctg ccagtatgat ttaaggtcgc 1921 aagtaattta ccctgaatcc tactatttta caagaaggaa atacttgctg aaagcccttt 1981 ttaaagcctt aaagagactc aagtctctga gagaccagtt ttcctttgca gaaaatctat 2041 accagataat cggtatagat tgctttcaga agaatgataa aaagatgttt aaatcttgtc 2101 gaaggctcac ctgatggaaa atggactggg ctactgagat atttttcatt atatatttga 2161 taacattctc taattctgtg aaaatatttc tttgaaaact ttgcaagtta agcaacttaa 2221 tgtgatgttg gataattggg ttttgtctat tttcacttct ccctaaataa tcttcacaga 2281 tattgtttga gggatattag gaaaattaat ttgttaactc gtctgtgcac agtattattt 2341 actctgtctg tagttcctga ataaattttc ttccatgctt gaactgggaa aattgcaaca 2401 cttttattct taatgacaac agtgaaaatc tcccagcata tacctagaaa acaattataa 2461 cttacaaaag attatccttg atgaaactca gaatttccac agtgggaatg aataagaagg 2521 caaaactcat
[0073] By "SLFN12 polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at NCBI Ref No. NP_060512.3 that interacts with PDE3A when bound to DNMDP and other complex inducing compounds. An exemplary human SLFN12 amino acid sequence is:
TABLE-US-00007 (SEQ ID NO: 7) MNISVDLETNYAELVLDVGRVTLGENSRKKMKDCKLRKKQNESVSRAMCAL LNSGGGVIKAEIENEDYSYTKDGIGLDLENSFSNILLFVPEYLDFMQNGNY FLIFVKSWSLNTSGLRITTLSSNLYKRDITSAKVMNATAALEFLKDMKKTR GRLYLRPELLAKRPCVDIQEENNMKALAGVFFDRTELDRKEKLTFTESTHV EIKNFSTEKLLQRIKEILPQYVSAFANTDGGYLFIGLNEDKEIIGFKAEMS DLDDLEREIEKSIRKMPVHHFCMEKKKINYSCKFLGVYDKGSLCGYVCALR VERFCCAVFAKEPDSWHVKDNRVMQLTRKEWIQFMVEAEPKFSSSYEEVIS QINTSLPAPHSWPLLEWQRQRHHCPGLSGRITYTPENLCRKLFLQHEGLKQ LICEEMDSVRKGSLIFSRSWSVDLGLQENHKVLCDALLISQDSPPVLYTFH MVQDEEFKGYSTQTALTLKQKLAKIGGYTKKVCVMTKIFYLSPEGMTSCQY DLRSQVIYPESYYFTRRKYLLKALFKALKRLKSLRDQFSFAENLYQIIGID CFQKNDKKMFKSCRRLT.
[0074] By "AIP polynucleotide" is meant any nucleic acid molecule encoding an AIP polypeptide or fragment thereof. An exemplary AIP nucleic acid sequence is provided at NCBI Ref: NM_003977.2:
TABLE-US-00008 (SEQ ID NO: 8) 1 ttttggcttc tgccctcaac caaaatggcg ctagctcgga agctgccgag gtgctaggag 61 ttgccgaagc aagtccggaa gctaccgagc gagtccggaa gttgccgaaa gggagcagcg 121 gggaaggagg atggcggata tcatcgcaag actccgggag gacgggatcc aaaaacgtgt 181 gatacaggaa ggccgaggag agctcccgga ctttcaagat gggaccaagg ttcgtgtcta 241 ccctaccctt ctccccctct gcggcgtggt gcgcatgcga ggcgggagga ggccttaggc 301 gagaggttgc gcatgcccag agggcagcgt ccactgcccc taccgctcac atgcagaact 361 cgacgctgat tgggctgaat ttaagtaggg ggtgaattcg ggcctgtctg ccccgccccc 421 tggctcggcc ttgtagcagc attggtgggg gaggccgtca gtcatcacaa gcgggttggg 481 gttgggggtt gatctcagtg cttgggcaga ccccacgctg gaggaaaccc agggccggga 541 gtggtcctcg ggtatctggg tttcaaggct catgatcctt tgtagatgga agggccttct 601 gaaaacactt agaccaactg ccgctgttta gagtggaaaa ccaagaccct gggacgtgca 661 aagccggaga acgggcccag aggtcaggtc tcccagacag ggactcttta gcagccttcc 721 tgctgcacta ggggcttgtt gggacagatg agggttggga agtaaagaac ctcccacttt 781 tctccttttt gccaggcccc cagatccagc ccctctgccc gcttctcccc caacctacaa 841 ctccaggctt ccctgcttct cctgtagttg cctcctcccg gagtgctttt cccagctgcc 901 acttgtttgc agagtaggga acctcccagg ggcagcccct gtgcccagca gagcagtcag 961 gcaggacatg cacattgagc aaatgagcac atgccccctg gccagcaccg tgccgaatcg 1021 ggcagctaag catcctagcc cagtgcagta taagtgccct gagagcagag gggagctgca 1081 tggctggagt gatccgctgt atgaaaagat atcttctcta agaagagaca ggatgtgtgg 1141 tgtgggttca tgcccccatg tgctgggggg ttggtggcgt tggaagaagg ggctggcaag 1201 ggggatcctg gatggaacag acatcagaag gagagatgtg aacaatggca ccccaagatc 1261 agaaacaggt ggtgttaaat aaccaatcgc cagcactgat tgagtgctca ctattcgaac 1321 attgtgctac atgcttcaca cgtttatttc ctacaatgtg agataggtac tgttgttgat 1381 tccgttttac cgatgtggaa actgacttca gagatgcagc atggtgcggc agttaagagc 1441 gtgggctcct ctaaccatat cctgtcgaga gttcaatctc caaacctctt ttctctgcac 1501 ccacccccag tgttatctct aaaaactctc cctgcccgga ttactcccag atgcagctct 1561 ccagtcatta actgtctctt aaacctgata tatagctccc tactcaccat atccacctgg 1621 aagcctggtt ggcaactcac acttaacctg ctccacctga ggcttctccg tgtcagggga 1681 accaacaacc ttcccgttgt tcagggcaaa aaccttagca tctctgtggt cctcccagtc 1741 tcacatccaa catcacatcc tcaatatcca gccaggatct gagttctcac cacttctgcc 1801 atcactgctt gggtccaggc catcctcatc tccagcctgg gttactgcag cgacctctaa 1861 ctctcctgcc tcttttgtcc ctctgtggtc tgttctcgtc ccagcagccg agcccatgcc 1921 agattcaatt ccttttttgc tcggagccac tcagtggctt ccatcacaga gtgaaaaaca 1981 gaggcctcac catagcctac aggccctgtg aggtccaccc ctactgacct gggtgagctc 2041 ccctgctgac cctgtggtgt accccacccc ctccttcact ctgctctgcc acactggcat 2101 tgctgctctt gaacacatca tgcatttgaa acgggaagtt cccttgtctc cctcgcaggg 2161 cgtgcgatgg gggagtggct cgcttcttca gtgccccgct gctcagacct ctgggggagc 2221 atacagatgg gcaggctgtg ggctccgacc tcatggcagt gtctaggggt gaatatttac 2281 agctccgtgt gttctagggt gctcttttag tttgtctatg ggaggcttgt gttaaccagc 2341 tcaattagac ccccttcctt atcacaagga cagagggctt tctgtagtct ggggttttct 2401 tgccttgatg tactggagta ctggagaatt agatcacttg tgggcttgga gaatgattgc 2461 aaattttttt ttatttttta ttttattttt tttttctgag atggagtttc actcttgttg 2521 cccaggctgg agtgcaatgg cacaatctct gcctcccagg ttcaagcaat tctcctgcct 2581 cagcctccca agtagctgag attacatgtg cctgacacca ggcccggcta atttttaaaa 2641 atgtttttag tagagatggg cttttaccat gttggccaag ctggtttcaa acgccttttt 2701 tttttttttt ttttttgaga cggagtcttg ctctattgcc caagctggag tgcagtggca 2761 tgatctcggt tcactgcaac ctccaccttc tgggttcaag tgattctcct gcctcagcct 2821 cccaagtagc tgggattaca ggcacccgcc atcatggcca gctaattttt gtatttttag 2881 tagagacggg gttttgccac attggccagg ctggtcttga actcctgacc tcaggtgatc 2941 cacccgcctt ggagatggtc ttcccctggg gttgggccac ttggtggccc cacctctcct 3001 ctgactgccc cagccaaact ccgcctcttc ctgccagttg atgacctgcc agcgtgcagg 3061 tgcctgtcag tgtgatcttc tgcttcttgc tcccctgaca tcctctcaat gaccaggagc 3121 tcgtcttctg ctgatgggct cctctgacat ctggctgcct gtgggtctac cccctagggg 3181 tgttgggttt ttataggcac aggatagggg tgtggcaggc cagggtggtc ttgggaaatg 3241 caacatttgg gcaggaaatg cctgttctca cctaggtctg tgggggtgga accctaccca 3301 gggaccacgc cctcctctac ccagcacttc ccttctcccc ttccaaatta tttaacagga 3361 ccatgctcct cccttcccag cacttccata tcacattgtc ccactgcaag gcttttttac 3421 acatgctgtt cttttggcct agaaagttcc tatcccaggg tccacttggc ttgctttctt 3481 ccttactccc caacccccca ctctgtttaa tccagcccca accctcttgc cctgctgttt 3541 cccaagcacg tggcttcacc tgccatgaca tattgttttg tttgatgccc atctcctccc 3601 tctagaagcg ccatgtgagc tccagggggg cagggacttt tttgtgtttt gcttgctgcc 3661 atgttctggt gtctagcaca gagcttgggc acatagtagg tgcttagtaa atatctgttg 3721 aggaatgact ggagtcagac tgcttggact cttgttccca ctcagccacc cactagccgt 3781 gtggcttggg cctattcctc ccctccttgt ggctttgttt tctcaccagc gtgggaggat 3841 gaagccaggt gtaaggtcag gtggtgtccc cggggaagcc ccgtccctta tgccgtctgc 3901 aggccgggga ctggacttct ccttgggggt cagggtgagg gtttgtgcct ttgcctgacc 3961 tcgcatgtgg cccacaggcc acgttccact accggacgct gcacagtgac gacgagggca 4021 ccgtgctgga cgacagccgg gctcgtggca agcccatgga gctcatcatt ggcaagaagt 4081 tcaagctgcc tgtgtgggag accatcgtgt gcaccatgcg agaaggggag attgcccagt 4141 tcctctgtga catcaaggtg tctgtcctgt acctgtctgc ggtggctgtc cagccaagcc 4201 ctattcctat tccctatccc cagggctcct cctccctcca ccctctgcta gactgccacc 4261 cgctttcttt ttttttttga gatggagtct tgctctgtcg cccaggctgg agtgcagtgg 4321 tacgatctca gctcactgca ccctccacct cctgggttca agcgattctt ctgcctcagc 4381 ctcccgagta gctgggatta caaacacccg ccatgatgcc tggctaattt ttgtattttt 4441 agtagagaca gggtttcacc atgttggcca ggctggtctt gaacacctga cctcaggtga 4501 tccacccgcc ttggcctccc aaagtgctgg gattataggc gtgtgccacc gcgcccggcc 4561 cacccactct ttccagacca ccacaccagc ctgctgatgg cgtcctggcc tccattccgc 4621 cttcccctat tagccagact gaggccaggg gactcgttct caaatgcaaa tgacctgtac 4681 atccctttgt ttcaaacctc tatgactcct ggtcactgta aggatagagc acagggggtc 4741 ctcacttcat gttgctgata cattcttgga aactgtgact aagagaaaaa acatacatca 4801 ggttttttct cagccaccgt catttctctc agcaaaattt tgttagaaca ttgatgagaa 4861 gaaaaattgg tttcgttatg tattgtttcg cctacagtca cagtttccaa gaacctactt 4921 aggacgttaa gtgaggactt aaaccgtata agctatagct gctcacatag ctttttgggg 4981 gctggcccct gccgtctcac cctcttactc aacctccctg cttttccttt ccattcccct 5041 tcttagccaa gatcttccct cttccttcaa agcttattcc tgggtcacca cctctaggaa 5101 gccctccctg actgctagtg gttggctcaa ctcccatgtt tgggtcctcc aaccctcatg 5161 ccctgcatgg ccaggatctg ctcttctgcc ttgtcctagg ctattgcaga gcagggatct 5221 ggcctgttta cttctagctt tgggatgccc agcgcgagcc agtccagagc caagactcag 5281 gaaatgcccg ctgatggcag cccggcagtc agcccctgtc cagacaacag ggcagtggga 5341 ggagtgggga ggacccgggt aggaggaatc tggttatctg gttcccacca gcctagcagc 5401 tttgccaagc aagagattag aggctaggtc ccctatgcct gtctccctgt ggggtttttt 5461 tttttttgac taagtctcac tctgttgccc aggctggagt gcagtggcgt gatcttggct 5521 cactgcaacc accatctcct gggttcagct gattctctgc cttagctgcc tgagtagctg 5581 ggattacagg cacctgccat cgtgcccggc tcatttttgt attttagcag agacgcggtt 5641 tcaccatgtt ggtcaggctg gtcttgaact cctgacctca ggtgatccgc ccgccttggc 5701 ctcccgaagt gctaggatta caggcgtgag ccaccacatc cggcctccct gagggttttg 5761 aagtggctgg cctgggccca gctctgaggt aggccctcag tggggtgtgg gtggggcaga 5821 aggaggagct gctgggaaca gaatgtgggg ggccccagtt ctttgcatag tccagcaaag 5881 ggccttatcc tctggaggga gaggaggtaa gaattctact gggcctgtaa ggaccaggga 5941 gacaggggtt gatggtaggc atgtgtctgt ggtgggggtg aggagggggt taggtgctct 6001 gtttggtggc cagagaatgt ggcagaagct ggggcttcac caggagagag ggctgagcga 6061 ctggaggagt cctgaattaa aagcctcctg tgcttaaacg gagtagggtc ccagttgtca 6121 ctctctgggc cttggtgttt gttctcagat ggtggtgggg aagggggctg ggccttgtgg 6181 acccggtgac cagccagccc acggtgacag agcccccggc gcccttgcct tcccgcagca 6241 tgtggtcctg tacccgctgg tggccaagag tctccgcaac atcgcggtgg gcaaggaccc 6301 cctggagggc cagcggcact gctgcggtgt tgcacagatg cgtgaacaca gctccctggg 6361 ccatgctgac ctggacgccc tgcagcagaa cccccagccc ctcatcttcc acatggagat 6421 gctgaaggtg aggggccacc gcgcctggtc tcaccaggcc cccactgccc agcctcaggg 6481 cggcgctggc ctgtccaccc aggggtggtg ggatccgcag gtggactgct gggggagcgg 6541 acagagacaa gaaaacctgt gcaggaccct tggcagtacc ctgggtctcc tttcctcctc 6601 cttcacatct caaatgtcac ctcctccagg aaaccggccc tgcccacccg gtctcctcat 6661 tctctgtctc gcagcagctc atttccttta tagcctctgc cgcaccttga agtcccttgg 6721 aattcatgga tttccttgtc catttaggga acctgccatg cagcatgatc tctgcgaggg 6781 cagggctttt caccgtcttg ttcactgttc tattcttagc acttggcaca gtgctgggca 6841 cacaggagat gtgacatcga tgtttgatgc tttttgagtg acaagtagct ctgctgctgg 6901 tgtgtgatgt ctgggggccc agccagccca gatgtgggtc aggtctgctg ctgacggacg 6961 cagctgtggt gtccccgagc cccgctgtga tatgccccat gccctgcagg tggagagccc 7021 tggcacgtac cagcaggacc catgggccat gacagacgaa gagaaggcaa aggcagtgcc 7081 acttatccac caggagggca accggttgta ccgcgagggg catgtgaagg aggctgctgc 7141 caagtactac gatgccattg cctgcctcaa gaacctgcag atgaaggtac tgcctggagg 7201 ctgaggggga ggatggatgg aggggggtgt ggagccaggg ggcccaggtc tacagcttct 7261 ccccgctccc tgcccccata ctcccaggaa cagcctgggt cccctgaatg gatccagctg 7321 gaccagcaga tcacgccgct gctgctcaac tactgccagt gcaagctggt ggtcgaggag 7381 tactacgagg tgctggacca ctgctcttcc atcctcaaca agtacgacgg tgagcaccgg 7441 gccctgggct gccgggggct gcgagtggtc agagagtggc ctttctcctg tcactgctgg
7501 ggtcaagacc tagcctttca caacccccat tctgagctcc cacgggggcc tgactaaatg 7561 cctctactcg gcagggctgt gggccccatt gtgccaatga agcatgaatg gtgtattggg 7621 ggtggggtgg catcctcagg tcagggaggg ctctctctcc cctgtgggcc catggtgcca 7681 ggagacatga gggcaggcag ctggccagga tcccccctca tgcccttgca tgcccactgc 7741 ccactggcct cccctgcaga caacgtcaag gcctacttca agcggggcaa ggcccacgcg 7801 gccgtgtgga atgcccagga ggcccaggct gactttgcca aagtgctgga gctggaccca 7861 gccctggcgc ctgtggtgag ccgagagctg caggccctgg aggcacggat ccggcagaag 7921 gacgaagagg acaaagcccg gttccggggg atcttctccc attgacagga gcacttggcc 7981 ctgccttacc tgccaagccc actgctgcag ctgccagccc ccctgcccgt gctgcgtcat 8041 gcttctgtgt atataaaggc ctttatttat ctctctctga
[0075] By "AIP polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at NCBI Reference Sequence: NP_003968.2 that can bind the aryl hydrocarbon receptor. AIP polypeptides may regulate expression of many xenobiotic metabolizing enzymes and bind specifically to and inhibit the activity of hepatitis B virus. Three transcript variants encoding different isoforms have been found for this gene. An exemplary human AIP amino acid sequence is:
TABLE-US-00009 (SEQ ID NO: 9) MADIIARLREDGIQKRVIQEGRGELPDFQDGTKATFHYRTLHSDDEGTVLD DSRARGKPMELIIGKKFKLPVWETIVCTMREGEIAQFLCDIKHVVLYPLVA KSLRNIAVGKDPLEGQRHCCGVAQMREHSSLGHADLDALQQNPQPLIFHME MLKVESPGTYQQDPWAMTDEEKAKAVPLIHQEGNRLYREGHVKEAAAKYYD AIACLKNLQMKEQPGSPEWIQLDQQITPLLLNYCQCKLVVEEYYEVLDHCS SILNKYDDNVKAYFKRGKAHAAVWNAQEAQADFAKVLELDPALAPVVSREL QALEARIRQKDEEDKARFRGIFSH.
[0076] By "TRRAP polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at NCBI Reference Sequence: NP_001231509.1 having histone acetyltransferase complex recruiting activity. An exemplary human TRRAP amino acid sequence is (which encodes the longer isoform):
TABLE-US-00010 (SEQ ID NO: 11) MAFVATQGATVVDQTTLMKKYLQFVAALTDVNTPDETKLKMMQEVSENFEN VTSSPQYSTFLEHIIPRFLTFLQDGEVQFLQEKPAQQLRKLVLEIIHRIPT NEHLRPHTKNVLSVMFRFLETENEENVLICLRIIIELHKQFRPPITQEIHE IFLDFVKQIYKELPKVVNRYFENPQVIPENTVPPPEMVGMITTIAVKVNPE REDSETRTHSIIPRGSLSLKVLAELPIIVVLMYQLYKLNIHNVVAEFVPLI MNTIAIQVSAQARQHKLYNKELYADFIAAQIKTLSFLAYIIRIYQELVTKY SQQMVKGMLQLLSNCPAETAHLRKELLIAAKHILTTELRNQFIPCMDKLFD ESILIGSGYTARETLRPLAYSTLADLVHHVRQHLPLSDLSLAVQLFAKNID DESLPSSIQTMSCKLLLNLVDCIRSKSEQESGNGRDVLMRMLEVFVLKFHT IARYQLSAIFKKCKPQSELGAVEAALPGVPTAPAAPGPAPSPAPVPAPPPP PPPPPPATPVTPAPVPPFEKQGEKDKEDKQTFQVTDCRSLVKTLVCGVKTI TWGITSCKAPGEAQFIPNKQLQPKETQIYIKLVKYAMQALDIYQVQIAGNG QTYIRVANCQTVRMKEEKEVLEHFAGVFTMMNPLTFKEIFQTTVPYMVERI SKNYALQIVANSFLANPTTSALEATILVEYLLDRLPEMGSNVELSNLYLKL FKLVFGSVSLFAAENEQMLKPHLHKIVNSSMELAQTAKEPYNYFLLLRALF RSIGGGSHDLLYQEFLPLLPNLLQGLNMLQSGLHKQHMKDLFVELCLTVPV RLSSLLPYLPMLMDPLVSALNGSQTLVSQGLRTLELCVDNLQPDFLYDHIQ PVRAELMQALWRTLRNPADSISHVAYRVLGKFGGSNRKMLKESQKLHYVVT EVQGPSITVEFSDCKASLQLPMEKAIETALDCLKSANTEPYYRRQAWEVIK CELVAMMSLEDNKHALYQLLAHPNETEKTIPNVIISHRYKAQDTPARKTFE QALTGAFMSAVIKDLRPSALPFVASLIRHYTMVAVAQQCGPFLLPCYQVGS QPSTAMEHSEENGSKGMDPLVLIDAIAICMAYEEKELCKIGEVALAVIFDV ASIILGSKERACQLPLFSYIVERLCACCYEQAWYAKLGGVVSIKFLMERLP LTWVLQNQQTELKALLEVMMDLTGEVSNGAVAMAKTTLEQLLMRCATPLKD EERAEEIVAAQEKSFHHVTHDLVREVTSPNSTVRKQAMHSLQVLAQVTGKS VTVIMEPHKEVLQDMVPPKKHLLRHQPANAQIGLMEGNTFCTTLQPRLFTM DLNVVEHKVFYTELLNLCEAEDSALTKLPCYKSLPSLVPLRIAALNALAAC NYLPQSREKIIAALFKALNSTNSELQEAGEACMRKFLEGATIEVDQIHTHM RPLLMMLGDYRSLTLNVVNRLTSVTRLFPNSENDKECDQMMQHLRKWMEVV VITHKGGQRSDGNESISECGRCPLSPFCQFEEMKICSAIINLFHLIPAAPQ TLVKPLLEVVMKTERAMLIEAGSPFREPLIKFLTRHPSQTVELFMMEATLN DPQWSRMFMSFLKHKDARPLRDVLAANPNRFITLLLPGGAQTAVRPGSPST STMRLDLQFQAIKIISIIVKNDDSWLASQHSLVSQLRRVWVSENFQERHRK ENMAATNWKEPKLLAYCLLNYCKRNYGDIELLFQLLRAFTGRFLCNMTFLK EYMEEEIPKNYSIAQKRALFFRFVDFNDPNFGDELKAKVLQHILNPAFLYS FEKGEGEQLLGPPNPEGDNPESITSVFITKVLDPEKQADMLDSLRIYLLQY ATLLVEHAPHHIHDNNKNRNSKLRRLMTFAWPCLLSKACVDPACKYSGHLL LAHIIAKFAIHKKIVLQVFHSLLKAHAMEARAIVRQAMAILTPAVPARMED GHQMLTHWTRKIIVEEGHTVPQLVHILHLIVQHFKVYYPVRHHLVQHMVSA MQRLGFTPSVTIEQRRLAVDLSEVVIKWELQRIKDQQPDSDMDPNSSGEGV NSVSSSIKRGLSVDSAQEVKRFRTATGAISAVFGRSQSLPGADSLLAKPID KQHTDTVVNFLIRVACQVNDNTNTAGSPGEVLSRRCVNLLKTALRPDMWPK SELKLQWFDKLLMTVEQPNQVNYGNICTGLEVLSFLLTVLQSPAILSSFKP LQRGIAACMTCGNTKVLRAVHSLLSRLMSIFPTEPSTSSVASKYEELECLY AAVGKVIYEGLTNYEKATNANPSQLFGTLMILKSACSNNPSYIDRLISVFM RSLQKMVREHLNPQAASGSTEATSGTSELVMLSLELVKTRLAVMSMEMRKN FIQAILTSLIEKSPDAKILRAVVKIVEEWVKNNSPMAANQTPTLREKSILL VKMMTYIEKRFPEDLELNAQFLDLVNYVYRDETLSGSELTAKLEPAFLSGL RCAQPLIRAKFFEVFDNSMKRRVYERLLYVTCSQNWEAMGNHFWIKQCIEL LLAVCEKSTPIGTSCQGAMLPSITNVINLADSHDRAAFAMVTHVKQEPRER ENSESKEEDVEIDIELAPGDQTSTPKTKELSEKDIGNQLHMLTNRHDKFLD TLREVKTGALLSAFVQLCHISTTLAEKTWVQLFPRLWKILSDRQQHALAGE ISPFLCSGSHQVQRDCQPSALNCFVEAMSQCVPPIPIRPCVLKYLGKTHNL WFRSTLMLEHQAFEKGLSLQIKPKQTTEFYEQESITPPQQEILDSLAELYS LLQEEDMWAGLWQKRCKYSETATAIAYEQHGFFEQAQESYEKAMDKAKKEH ERSNASPAIFPEYQLWEDHWIRCSKELNQWEALTEYGQSKGHINPYLVLEC AWRVSNWTAMKEALVQVEVSCPKEMAWKVNMYRGYLAICHPEEQQLSFIER LVEMASSLAIREWRRLPHVVSHVHTPLLQAAQQIIELQEAAQINAGLQPTN LGRNNSLHDMKTVVKTWRNRLPIVSDDLSHWSSIFMWRQHHYQGKPTWSGM HSSSIVTAYENSSQHDPSSNNAMLGVHASASAIIQYGKIARKQGLVNALDI LSRIHTIPTVPIVDCFQKIRQQVKCYLQLAGVMGKNECMQGLEVIESTNLK YFTKEMTAEFYALKGMFLAQINKSEEANKAFSAAVQMHDVLVKAWAMWGDY LENIFVKERQLHLGVSAITCYLHACRHQNESKSRKYLAKVLWLLSFDDDKN TLADAVDKYCIGVPPIQWLAWIPQLLTCLVGSEGKLLLNLISQVGRVYPQA VYFPIRTLYLTLKIEQRERYKSDPGPIRATAPMWRCSRIMHMQRELHPTLL SSLEGIVDQMVWFRENWHEEVLRQLQQGLAKCYSVAFEKSGAVSDAKITPH TLNFVKKLVSTFGVGLENVSNVSTMFSSAASESLARRAQATAQDPVFQKLK GQFTTDFDFSVPGSMKLHNLISKLKKWIKILEAKTKQLPKFFLIEEKCRFL SNFSAQTAEVEIPGEFLMPKPTHYYIKIARFMPRVEIVQKHNTAARRLYIR GHNGKIYPYLVMNDACLTESRREERVLQLLRLLNPCLEKRKETTKRHLFFT VPRVVAVSPQMRLVEDNPSSLSLVEIYKQRCAKKGIEHDNPISRYYDRLAT VQARGTQASHQVLRDILKEVQSNMVPRSMLKEWALHTFPNATDYWTFRKMF TIQLALIGFAEFVLHLNRLNPEMLQIAQDTGKLNVAYFRFDINDATGDLDA NRPVPFRLTPNISEFLTTIGVSGPLTASMIAVARCFAQPNFKVDGILKTVL RDEIIAWHKKTQEDTSSPLSAAGQPENMDSQQLVSLVQKAVTAIIVITRLH NLAQFEGGESKVNTLVAAANSLDNLCRMDPAWHPWL
[0077] By "TRRAP polynucleotide" is meant any nucleic acid molecule encoding an TRRAP polypeptide or fragment thereof. An exemplary TRRAP nucleic acid sequence is provided at NCBI Ref: NM_001244580.1:
TABLE-US-00011 (SEQ ID NO: 10) 1 cgcgccgggg cctggtgctc ggtcggcggg tgctgccgct ttaagcgggg gcgggactgc 61 gcgcggccga gcggttgcga cgagggctcg gctgggggtc gccggggtcg cgggccgggc 121 ctgcaggagc cgggccgccg aggtcggggc tggttgaact catggacctg atacttttct 181 cttgagaagc aaaccagccc aaaagaaaaa tggcgtttgt tgcaacacag ggggccacgg 241 tggttgacca gaccactttg atgaaaaagt accttcagtt tgtggcagct ctcacagatg 301 tgaatacacc tgatgaaaca aagttgaaaa tgatgcaaga agttagtgaa aattttgaga 361 atgtcacgtc atctcctcag tattctacat tcctagaaca tatcatccct cgattcctta 421 catttctcca agatggagaa gttcagtttc ttcaggagaa accagcacag caactgcgga 481 agctcgtact tgaaataatt catagaatac caaccaacga acatcttcgt cctcacacaa 541 aaaatgtttt gtctgtgatg tttcgctttt tagagacgga aaatgaagaa aatgttctta 601 tttgtctaag aataattatt gagctacaca aacagttcag gccaccgatc acacaagaaa 661 ttcatcattt tctggatttt gtgaaacaga tttacaagga gcttccaaaa gtagtgaacc 721 gctactttga gaaccctcaa gtgatccccg agaacacagt gcctccccca gaaatggttg 781 gtatgataac aacgattgct gtgaaagtca acccggagcg tgaggacagt gagactcgaa 841 cacattccat cattccgagg ggatcacttt ctctgaaagt gttggcagaa ttgcccatta 901 ttgttgtttt aatgtatcag ctctacaaac tgaacatcca caatgttgtt gctgagtttg 961 tgcccttgat catgaacacc attgccattc aggtgtctgc acaagcgagg caacataagc 1021 tttacaacaa ggagttgtat gctgacttca ttgctgctca gattaaaaca ttgtcatttt 1081 tagcttacat tatcaggatt taccaggagt tggtgactaa gtattctcag cagatggtga 1141 aaggaatgct ccagttactt tcaaattgtc cagcagagac tgcacacctc agaaaggagc 1201 ttctgattgc tgccaaacac atcctcacca cagagctgag aaaccagttc attccttgca 1261 tggacaagct gtttgatgaa tccatactaa ttggctcagg atatactgcc agagagactc 1321 taaggcccct cgcctacagc acgctggccg acctcgtgca ccatgtccgc cagcacctgc 1381 ccctcagcga cctctccctc gccgtccagc tcttcgccaa gaacatcgac gatgagtccc 1441 tgcccagcag catccagacc atgtcctgca agctcctgct gaacctggtg gactgcatcc 1501 gttccaagag cgagcaggag agtggcaatg ggagagacgt cctgatgcgg atgctggagg 1561 ttttcgttct caaattccac acaattgctc ggtaccagct ctctgccatt tttaagaagt 1621 gtaagcctca gtcagaactt ggagccgtgg aagcagctct gcctggggtg cccactgccc 1681 ctgcagctcc tggccctgct ccctccccag cccctgtccc tgccccacct ccacccccgc 1741 ccccaccccc acctgccacc cctgtgaccc cggcccccgt gcctcccttc gagaagcaag 1801 gagaaaagga caaggaagac aagcagacat tccaagtcac agactgtcga agtttggtca 1861 aaaccttggt gtgtggtgtc aagacaatca cgtggggcat aacatcatgc aaagcacctg 1921 gtgaagctca gttcattccc aacaagcagt tacaacccaa agagacacag atttacatca 1981 aacttgtgaa atatgcaatg caagctttag atatttatca ggtccagata gcaggaaatg 2041 gacagacata catccgtgtg gccaactgcc agactgtgag aatgaaagag gagaaggagg 2101 tattggagca tttcgctggt gtgttcacaa tgatgaaccc cttaacgttc aaagaaatct 2161 tccaaactac ggtcccttat atggtggaga gaatctcaaa aaattatgct cttcagattg 2221 ttgccaattc cttcttggca aatcctacta cctctgctct gtttgctacg attctggtgg 2281 aatatctcct tgatcgcctg ccagaaatgg gctccaacgt ggagctctcc aacctgtacc 2341 tcaagctgtt caagctggtc tttggctctg tctccctctt tgcagctgaa aatgaacaaa 2401 tgctgaagcc tcacttgcac aagattgtga acagctctat ggagctcgcg cagactgcca 2461 aggaacccta caactacttc ttgctgctac gggcgctgtt tcgctctatt ggtggaggta 2521 gccacgatct cttgtatcag gagttcttgc ctctccttcc aaacctcctg caagggctga 2581 acatgcttca gagtggcctg cacaagcagc acatgaagga cctctttgtg gagctgtgtc 2641 tcaccgtccc tgtgcggctg agctcgcttt tgccgtacct gcccatgctt atggatccct 2701 tggtgtctgc actcaatggg tctcagacat tggtcagcca aggcctcagg acgctggagc 2761 tgtgtgtgga caacctgcag cccgacttcc tctacgacca catccagccg gtgcgcgcag 2821 agctcatgca ggctctgtgg cgcaccttac gcaaccctgc tgacagcatc tcccacgtgg 2881 cctaccgtgt gctcggtaag tttggcggca gtaacaggaa gatgctgaag gagtcgcaga 2941 agctgcacta cgttgtgacc gaggttcagg gccccagcat cactgtggag ttttccgact 3001 gcaaagcttc tctccagctc cccatggaga aggccattga aactgctctg gactgcctga 3061 aaagcgccaa cactgagccc tactaccgga ggcaggcgtg ggaagtgatc aaatgcttcc 3121 tggtggccat gatgagcctg gaggacaaca agcacgcact ctaccagctc ctggcacacc 3181 ccaactttac agaaaagacc atccccaatg ttatcatctc acatcgctac aaagcccagg 3241 acactccagc ccggaagact tttgagcagg ccctgacagg cgccttcatg tctgctgtca 3301 ttaaggacct gcggcccagc gccctgccct ttgtcgccag cttgatccgc cactatacga 3361 tggtggcagt cgcccagcag tgtggccctt tcttgctgcc ttgctaccag gtgggcagcc 3421 agcccagcac agccatgttt cacagtgaag aaaatggctc gaaaggaatg gatcctttgg 3481 ttctcattga tgcaattgct atttgtatgg catatgaaga aaaggagctt tgcaaaatcg 3541 gggaggtggc cctagctgtg atatttgatg ttgcaagtat catcctgggc tccaaggaga 3601 gggcctgcca gctgcccctg ttttcttaca tcgtggagcg cctgtgtgca tgttgttatg 3661 aacaggcgtg gtatgcaaag ctggggggtg tggtgtctat taagtttctc atggagcggc 3721 tgcctctcac ttgggttctc cagaaccagc agacattcct gaaagcactt ctctttgtca 3781 tgatggactt aactggagag gtttccaatg gggcagtcgc tatggcaaag accacgctgg 3841 agcagcttct gatgcggtgc gcaacgcctt taaaagacga ggagagagcc gaagagatcg 3901 tggccgccca ggaaaagtct ttccaccatg tgacacacga cttggttcga gaagtcacct 3961 ctccaaactc cactgtgagg aagcaggcca tgcattcgct gcaggtgttg gcccaggtca 4021 ctgggaagag tgtcacggtg atcatggaac cccacaaaga ggtcctgcag gatatggtcc 4081 cccctaagaa gcacctgctc cgacaccagc ctgccaacgc acagattggc ctgatggagg 4141 ggaacacgtt ctgtaccacg ttgcagccca ggctcttcac aatggacctt aacgtggtgg 4201 agcataaggt gttctacaca gagctgttga atttgtgtga ggctgaagat tcagctttaa 4261 caaagctgcc ctgttataaa agccttccgt cactcgtacc tttacgaatt gcggcattaa 4321 atgcacttgc tgcctgcaat taccttcctc agtccaggga gaaaatcatc gctgcactct 4381 tcaaagccct gaattccacc aatagtgagc tccaagaggc cggagaagcc tgtatgagaa 4441 agtttttaga aggtgctacc atagaagtcg atcaaatcca cacacatatg cgacctttgc 4501 tgatgatgct gggagattac cggagcttga cgctgaatgt tgtgaatcgc ctgacttcgg 4561 tcacgaggct cttcccaaat tccttcaatg ataaattttg tgatcagatg atgcaacatc 4621 tgcgcaagtg gatggaagtg gtggtgatca cccacaaagg gggccagagg agcgacggaa 4681 acgaaagcat ttccgagtgc gggagatgtc ccttgtctcc attctgtcag tttgaggaaa 4741 tgaagatttg ctcagcaatt ataaaccttt ttcatctgat cccggctgct cctcagacac 4801 tggtgaagcc tttgctagag gttgtcatga aaacggagcg ggcgatgctg atcgaggcgg 4861 ggagtccatt ccgagagccc ctgatcaagt tcctgactcg acatccctcg cagacagtgg 4921 agctgttcat gatggaagcc acactgaacg atccccagtg gagcagaatg tttatgagtt 4981 ttttaaaaca caaagacgcc agacctctgc gggatgtgct ggctgccaac cccaacaggt 5041 tcatcaccct gctgctgccg gggggtgccc agacggctgt gcgccccggt tcgcccagca 5101 ccagcaccat gcgcctggac ctccagttcc aggccatcaa gatcataagc attatagtga 5161 aaaacgatga ctcctggctg gccagccagc actctctggt gagccagttg cgacgtgtgt 5221 gggtgagtga gaacttccaa gagaggcacc gcaaggagaa catggcagcc accaactgga 5281 aggagcccaa gctgctggcc tactgcctgc tgaactactg caaaaggaat tacggagata 5341 tagaattgct gttccagctg ctccgagcct ttactggtcg ttttctctgc aacatgacat 5401 tcttaaaaga gtatatggag gaagagattc ccaaaaatta cagcatcgct cagaaacgtg 5461 ccctgttctt tcgctttgta gacttcaacg accccaactt cggagatgaa ttaaaagcta 5521 aagttctgca gcatatcttg aatcctgctt tcttgtacag ctttgagaag ggggaaggag 5581 agcagctctt gggacctccc aatccagaag gagataaccc agaaagcatc accagtgtgt 5641 ttattaccaa ggtcctggac cccgagaagc aggcggacat gctggactcg ctgcggatct 5701 acctgctgca gtacgccacg ctgctggtgg agcacgcccc ccaccacatc catgacaaca 5761 acaagaaccg caacagcaag ctgcgccgcc tcatgacctt cgcctggccc tgcctgctct 5821 ccaaggcctg cgtggaccca gcctgcaagt acagcggaca cttgctcctg gcgcacatta 5881 tcgccaaatt cgccatacac aagaagatcg tcctgcaggt ttttcatagt ctcctcaagg 5941 ctcacgcaat ggaagctcga gcgatcgtca gacaggcgat ggccattctg accccggcgg 6001 tgccggccag gatggaggac gggcaccaga tgctgaccca ctggacccgg aagatcattg 6061 tggaggaggg gcacaccgtc ccgcagctgg tccacattct gcacctgata gtgcaacact 6121 tcaaggtgta ctacccggta cggcaccact tggtgcagca catggtgagc gccatgcaga 6181 ggctgggctt cacgcccagt gtcaccatcg agcagaggcg gctggccgtg gacctgtctg 6241 aagtcgtcat caagtgggag ctgcagagga tcaaggacca gcagccggat tcagatatgg 6301 acccaaattc cagtggagaa ggagtcaatt ctgtctcatc ctccattaag agaggcctgt 6361 ccgtggattc tgcccaggaa gtgaaacgct ttaggacggc caccggagcc atcagtgcag 6421 tctttgggag gagccagtcg ctacctggag cagactctct cctcgccaag cccattgaca 6481 agcagcacac agacactgtg gtgaacttcc ttatccgcgt ggcctgtcag gttaatgaca 6541 acaccaacac agcggggtcc cctggggagg tgctctctcg ccggtgtgtg aaccttctga 6601 agactgcgtt gcggccagac atgtggccca agtccgaact caagctgcag tggttcgaca 6661 agctgctgat gactgtggag cagccaaacc aagtgaacta tgggaatatc tgcacgggcc 6721 tagaagtgct gagcttcctg ctaactgtcc tccagtcccc agccatcctc agtagcttca 6781 aacctctgca gcgtggaatt gccgcctgca tgacatgtgg aaacaccaag gtgttgcgag 6841 ccgtccacag ccttctctcg cgcctgatga gcattttccc aacagagccg agtacttcca 6901 gtgtggcctc caaatatgaa gagctggagt gcctctacgc agccgtcgga aaggtcatct 6961 atgaagggct caccaactac gagaaggcca ccaatgccaa tccctcccag ctcttcggga 7021 cccttatgat cctcaagtct gcctgcagca acaaccccag ctacatagac aggctgatct 7081 ccgtctttat gcgctccctg cagaagatgg tccgggagca tttaaaccct caggcagcgt 7141 caggaagcac cgaagccacc tcaggtacaa gcgagctggt gatgctgagt ctggagctgg 7201 tgaagacgcg cctggcagtg atgagcatgg agatgcggaa gaacttcatc caggccatcc 7261 tgacatccct catcgaaaaa tcaccagatg ccaaaatcct ccgggctgtg gtcaaaatcg 7321 tggaagaatg ggtcaagaat aactccccaa tggcagccaa tcagacacct acactccggg 7381 agaagtccat tttgcttgtg aagatgatga cttacataga aaaacgcttt ccggaagacc 7441 ttgaattaaa tgcccagttt ttagatcttg ttaactatgt ctacagggat gagaccctct
7501 ctggcagcga gctgacggcg aaacttgagc ctgcctttct ctctgggctg cgctgtgccc 7561 agccactcat cagggcaaag tttttcgagg tttttgacaa ctccatgaaa cgtcgtgtct 7621 acgagcgctt gctctatgtg acctgttcgc agaactggga agccatgggg aaccacttct 7681 ggatcaagca gtgcattgag ctgcttctgg ccgtgtgtga gaagagcacc cccattggca 7741 ccagctgcca aggagccatg ctcccgtcca tcaccaacgt catcaacctg gccgatagcc 7801 acgaccgtgc cgccttcgcc atggtcacac atgtcaagca ggagccccgg gagcgggaga 7861 acagcgagtc caaagaggag gatgtagaga tagacatcga actagctcct ggggatcaga 7921 ccagcacgcc caaaaccaaa gaactttcag aaaaggacat tggaaaccag ctgcacatgc 7981 taaccaacag gcacgacaag tttctggaca ctctccgaga ggtgaagact ggagcgctgc 8041 tcagcgcttt cgttcagctg tgccacattt ccacgacgct ggcagagaag acgtgggtcc 8101 agcttttccc cagattgtgg aagatcctct ctgacagaca gcagcatgca ctcgcgggtg 8161 agataagtcc atttctgtgc agcggcagtc accaggtgca gcgggactgc cagcccagcg 8221 cgctgaactg ctttgtggaa gccatgtccc agtgcgtgcc gccaatcccc atccgaccct 8281 gcgtcctgaa gtacctgggg aagacacaca acctctggtt ccggtccacg ctgatgttgg 8341 agcaccaggc ttttgaaaag ggtctgagtc ttcagattaa gccgaagcaa acaacggagt 8401 tttatgagca ggagagcatc accccgccgc agcaggagat actggattcc cttgcggagc 8461 tttactccct gttacaagag gaagatatgt gggctggtct gtggcagaag cggtgcaagt 8521 actcggagac agcgactgcg attgcttacg agcagcacgg gttctttgag caggcacaag 8581 aatcctatga aaaggcaatg gataaagcca aaaaagaaca tgagaggagt aacgcctccc 8641 ctgctatttt ccctgaatac cagctctggg aagaccactg gattcgatgc tccaaggaat 8701 tgaaccagtg ggaagccctg acggagtacg gtcagtccaa aggccacatc aacccctacc 8761 tcgtcctgga gtgcgcctgg cgggtgtcca actggactgc catgaaggag gcgctggtgc 8821 aggtggaagt gagctgtccg aaggagatgg cctggaaggt gaacatgtac cgcggatacc 8881 tggccatctg ccaccccgag gagcagcagc tcagcttcat cgagcgcctg gtggagatgg 8941 ccagcagcct ggccatccgc gagtggcggc ggctgcccca cgtagtgtcc cacgtgcaca 9001 cgcctctcct acaggcagcc cagcaaatca tcgaactcca ggaagctgca caaatcaacg 9061 caggcttaca gccaaccaac ctgggaagga acaacagcct gcacgacatg aagacggtgg 9121 tgaagacctg gaggaaccga ctgcccatcg tgtctgacga cttgtcccac tggagcagca 9181 tcttcatgtg gaggcagcat cattaccagg gtaaaccgac ctggtccggc atgcattcat 9241 catcgattgt aactgcctat gagaatagct ctcagcatga tcccagttca aataacgcta 9301 tgcttggggt tcatgcatca gcttcagcga tcatccagta tggaaaaatc gcccggaaac 9361 aaggactggt caatgtagct ctggatatat taagtcggat tcatactatt ccaactgttc 9421 ctatcgtgga ttgcttccag aagattcgac agcaagttaa atgctacctc cagctggcag 9481 gcgtcatggg caaaaacgag tgcatgcagg gccttgaagt tattgaatct acaaatttaa 9541 aatacttcac aaaagagatg acagccgaat tttatgcact gaagggaatg ttcttggctc 9601 agatcaacaa gtccgaggag gcaaacaaag ccttctctgc agctgtgcag atgcacgatg 9661 tgctggtgaa agcctgggcc atgtggggcg actacctgga gaacatcttt gtgaaggagc 9721 ggcagctgca cctgggcgtg tctgccatca cctgctacct gcacgcctgc cggcatcaga 9781 acgagagcaa atcgaggaaa tacttagcca aggtgctgtg gcttttgagt tttgatgatg 9841 acaaaaacac tttggcagat gccgtcgaca agtactgcat tggtgtgcca cccatccagt 9901 ggctggcctg gatcccacag ctgctcacct gcctggttgg ctcggaggga aagctgctct 9961 tgaacctcat tagccaggtt ggacgcgtgt atccccaagc ggtctacttt cccatccgga 10021 ccctgtacct gaccctgaaa atagaacagc gggaacgcta caagagcgat ccagggccca 10081 taagagcaac agcacccatg tggcgctgca gccgaatcat gcacatgcag cgagagctcc 10141 accccaccct tctgtcttcc ctggaaggca tcgtcgatca gatggtctgg ttcagagaaa 10201 attggcatga agaggttctc aggcagctcc aacagggcct ggcgaaatgt tactccgtgg 10261 cgtttgagaa aagtggagcg gtgtccgatg ctaaaatcac cccccacact ctcaattttg 10321 tgaagaagtt ggtgagcacg tttggggtgg gcctggagaa tgtgtccaac gtctcgacca 10381 tgttctccag cgcagcctct gagtctctgg cccggcgggc gcaggccact gcacaagacc 10441 ctgtctttca gaagctgaaa ggccagttca cgacggattt tgacttcagc gttccaggat 10501 ccatgaagct tcataatctt atttctaagt tgaaaaagtg gatcaaaatc ttggaggcca 10561 agaccaagca actccccaaa ttcttcctca tagaggaaaa gtgccggttc ttgagcaatt 10621 tctcggcaca gacagctgaa gtggaaattc ctggggagtt tctgatgcca aagccaacgc 10681 attattacat caagattgca cggttcatgc cccgggtaga gattgtgcag aagcacaaca 10741 ccgcagcccg gcggctgtac atccggggac acaatggcaa gatctaccca tacctcgtca 10801 tgaacgacgc ctgcctcaca gagtcacggc gagaggagcg tgtgttgcag ctgctgcgtc 10861 tgctgaaccc ctgtttggag aagagaaagg agaccaccaa gaggcacttg tttttcacag 10921 tgccccgggt tgtggcagtt tccccacaga tgcgcctcgt ggaggacaac ccctcttcac 10981 tttcccttgt ggagatctac aagcagcgct gcgccaagaa gggcatcgag catgacaacc 11041 ccatctcccg ttactatgac cggctggcta cggtgcaggc gcggggaacc caagccagcc 11101 accaggtcct ccgcgacatc ctcaaggagg ttcagagtaa catggtgccg cgcagcatgc 11161 tcaaggagtg ggcgctgcac accttcccca atgccacgga ctactggacg ttccggaaga 11221 tgttcaccat ccagctggct ctgataggct tcgcggaatt cgtcctgcat ttaaatagac 11281 tcaaccccga gatgttacag atcgctcagg acactggcaa actgaatgtt gcctactttc 11341 gatttgacat aaacgacgcg actggagacc tggatgccaa ccgtcctgtc ccatttcgac 11401 tcacgcccaa catttctgag tttctgacca ccatcggggt ctccggcccg ttgacagcgt 11461 ccatgattgc ggtcgcccgg tgcttcgccc agccaaactt taaggtggat ggcattctga 11521 aaacggttct ccgggacgag atcattgctt ggcacaaaaa aacacaagag gacacgtcct 11581 ctcctctctc ggccgccggg cagccagaga acatggacag ccagcaactg gtgtccctgg 11641 ttcagaaagc cgtcaccgcc atcatgaccc gcctgcacaa cctcgcccag ttcgaaggcg 11701 gggaaagcaa ggtgaacacc ctggtggccg cggcaaacag cctggacaat ctgtgccgca 11761 tggaccccgc ctggcacccc tggctgtgac tgtggccgcc acggccaccc ggaatgtgaa 11821 gggcgctccg ggctctgagc ccgcagcttt tacgacttct ccctgcctcg ttccttatat 11881 tcacagaagc cccatagttt cactgggttg cggttatttt cctggtagtt tgcgtgtaag 11941 aaagggagaa tatagtttta gaggaagctg aactatgacg atgctgggcg aagcggttgg 12001 aaatggcaga gctgaaactt attccaagct ttcaaaataa tcttttaaga agccaggatt 12061 ctccggtctg gaatttctga gtgagtcctt tttttatggt gtcctccctc tgtgaatgta 12121 caggcggaac tgtacgaaca gctcccttcc atccattttt aactctttcg gaaataacac 12181 ctcacagcag cttcgtgctt ttgtacagac ctttgtaaca agtgtacaga aaactcattt 12241 tgtttgagaa acaggagttg atgaacccat catgctggtt tttctctgag cacaaagttt 12301 taggctgtac acagccagcc ttgggaatct cgttgagcgt tcggcgtgga tccacggggc 12361 caggccaccc tgcgggagcg ccacacgcat ccacttcgga ttcagtgggt gaagacagaa 12421 ctctgagagt ctgcaggcgg ctcctgtgct ttttatttct ggctcttcgg atgtcttcta 12481 gacatttact atcactgcac ctgaagaaaa aatcactttt accttcctaa tttaaaaaga 12541 caaaacagaa atgtacgttc cttcgctagc tttagtcttt ctgttcccat ttttataaat 12601 ctgagcattg ataatgttct atctaaattt gtacagtgtg attttttttt ttagaataaa 12661 tattttataa aagggttaaa aaaaaaaaaa aaaaa
[0078] In certain implementations, the marker that detects polynucleotide may be the polynucleic region that encodes the protein.
[0079] By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods, such as those described herein. As used herein, an alteration includes at least a 10% change in expression levels, for example a 25% change, a 40% change, and a 50% or greater change in expression levels.
[0080] A "chemically induced" complex (e.g., chemically induced PDE3A-SLFN12 complex, chemically induced PDE3B-SLFN12 complex) is the complex formed by indicated agents following contact with an active compound, such as a PDE3A modulator or a PDE3B modulator. Typically, the active compounds described herein are a chemical compound inducing PDE3A-SLFN12 or PDE3B-SLFN12 complexes, such as e.g. DNMDP or a compound of WO2019/025562.
[0081] By "modulator" is meant any agent that binds to a polypeptide and alters a biological function or activity of the polypeptide. A modulator includes, without limitation, agents that increase binding of a polypeptide to another agent. For example, a modulator may promote binding of a polypeptide to another polypeptide. In some embodiments a modulator of PDE3A or PDE3B promotes binding of these proteins to SLFN12. In some embodiments, a modulator of PDE3A polypeptide is DNMDP. In other embodiments, a modulator of PDE3A is an exemplified compound of WO2019/025562.
[0082] The term "capture reagent" refers to a reagent, for example an antibody or antigen binding protein, capable of binding a target molecule or analyte to be detected in a biological sample. The capture reagent may be immobilized, for example on an assay surface, such as a solid substrate or reaction vessel. The capture reagents described herein may bind to one or more of PDE3A, PDE3B, SLFN12, AIP, and TRRAP.
[0083] "Detect" refers to identifying the presence, absence or amount of the analyte to be detected. In particular embodiments, the analyte is an AIP, TRRAP, PDE3A, PDE3B, or SLFN12 polypeptide.
[0084] By "effective amount" or "therapeutically effective amount" is meant the amount of a compound described herein required to ameliorate the symptoms (e.g., treat, prevent) of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present disclosure for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. In some embodiments, the compound is DNMDP or a compound of WO2019/025562.
[0085] The terms "healthy", "normal" and "non-neoplastic" are used interchangeably herein to refer to a subject or particular cell or tissue that is devoid (at least to the limit of detection) of a disease condition, such as a neoplasia. In some embodiments, the reference may be a healthy cell.
[0086] The disclosure provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein. In addition, the methods of the disclosure provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.
[0087] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
[0088] By "marker" or "biomarker" is meant any protein or polynucleotide having an alteration in expression level or activity relative to a reference that is associated with a disease or disorder, such as cancer. In particular embodiments, a marker of the disclosure is AIP (e.g., AlP polypeptide, AIP polynucleotide), TRRAP (e.g., TRRAP polypeptide, TRRAP polynucleotide), PDE3A (e.g., PDE3A polypeptide, PDE3A polynucleotide), PDE3B (e.g., PDE3B polypeptide, PDE3B polynucleotide), or SLFN12 (e.g., SLFN12 polypeptide, SLFN12 polynucleotide). In certain implementations, the marker may comprise portions of a polynucleotides sequence (e.g., SEQ ID NOS: 1, 4, 6, 8, 10) which encode the polypeptide (e.g., AIP polypeptide, TRRAP polypeptide, PDE3A polypeptide, PDE3B polypeptide, SLFN12 polypeptide). In some embodiments, the marker may have any one of SEQ ID NOS: 1-11. In some embodiments, a marker may comprise at least 75% or at least 80% or at least 85% sequence identity to SEQ ID NOS 2, 3, 5, 7, 9, or 11. In certain embodiments, the presence of a marker in a cancer cell identifies that cell as responsive to therapy.
[0089] Nucleic acid molecules (e.g., polynucleotides) useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having substantial identity to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Typically, when polynucleotides hybridize and at least one strand of a nucleic acid molecule hybridizes, they are able to pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency.
[0090] For example, "stringent" salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate or less than about 500 mM NaCl and 50 mM trisodium citrate or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide or at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30.degree. C. or at least about 37.degree. C. of at least about 42.degree. C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a specific embodiment, hybridization will occur at 30.degree. C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In some embodiments, hybridization will occur at 37.degree. C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In certain aspects, hybridization may occur at 42.degree. C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 .mu.g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
[0091] For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps may be less than about 30 mM NaCl and 3 mM trisodium citrate, or more less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25.degree. C., for example of at least about 42.degree. C., or at least about 68.degree. C. In some embodiments, wash steps will occur at 25.degree. C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In certain embodiments, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a specific implementation, wash steps may occur at 68.degree. C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
[0092] Typically, substantially identical polypeptides or nucleic acids exhibit at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). In certain implementations, such a sequence is at least 60%, or at least 80% or 85% or at least 90% or at least 95% or at least 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
[0093] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions may include substitutions within the following groups:
[0094] glycine, alanine;
[0095] valine, isoleucine, leucine;
[0096] aspartic acid, glutamic acid, asparagine, glutamine;
[0097] aspartic acid, glutamic acid;
[0098] asparagine, glutamine;
[0099] serine, threonine; lysine, arginine; and
[0100] phenylalanine, tyrosine.
In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e.sup.-3 and e.sup.-100 indicating a closely related sequence.
[0101] The term "pharmaceutical composition," as used herein, represents a composition containing a compound (e.g., a PDE3 modulator) formulated with a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of a disease in a mammal. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gelcap); for topical administration (e.g., as a cream, gel, lotion, ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); or in any other formulation described herein.
[0102] Useful pharmaceutical carriers for the preparation of the compositions hereof, can be solids, liquids, or gases. Thus, the compositions can take the form of tablets, pills, capsules, suppositories, powders, enterically coated or other protected formulations (e.g., binding on ion-exchange resins), sustained release formulations, solutions, suspensions, elixirs, and aerosols. The carrier can be selected from the various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, and sesame oil. Water, saline, aqueous dextrose, and glycols are liquid carriers, particularly (when isotonic with the blood) for injectable solutions. For example, formulations for intravenous administration comprise sterile aqueous solutions of the active compound(s) which are prepared by dissolving solid active compound(s) in water to produce an aqueous solution, and rendering the solution sterile. Suitable pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, talc, gelatin, malt, rice, flour, chalk, silica, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol. The compositions may be subjected to conventional pharmaceutical additives, such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, and buffers. Suitable pharmaceutical carriers and their formulation are described in Remington's Pharmaceutical Sciences by E. W. Martin. Such compositions will, in any event, contain an effective amount of the active compound together with a suitable carrier so as to prepare the proper dosage form for administration to the recipient.
[0103] As used herein, the term "pharmaceutically acceptable salt" refers to salts of any of the compounds mentioned herein that within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P. H. Stahl and C. G. Wermuth), Wiley-VCH, 2008. Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, dichloroacetate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glutamate, glycerophosphate, hemisulfate, heptonate, hexanoate, hippurate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, isethionate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, mucate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate salts. Representative basic salts include alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, aluminum salts, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, caffeine, and ethylamine.
[0104] As used herein, the term "subject" refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include any animal (e.g., mammals, such as mice, rats, rabbits, non-human primates, and humans). The subject may be domesticated animals (e.g., cows, calves, sheep, lambs, horses, foals, pigs, piglets), or animals in the family Muridae (e.g., rats, mice). A subject may seek or be in need of treatment, require treatment, be receiving treatment, may be receiving treatment in the future, or a human or animal that is under care by a trained professional for a particular disease or condition.
[0105] A "patient in need thereof" as used herein, refers to a human individual who may be identified as having a disease, disorder, or condition responsive to complex formation. As described herein, in some embodiments, an individual in need thereof is suffering from a proliferative disorder, such as cancer. In some embodiments, an individual in need thereof has been diagnosed by a medical doctor with a proliferative disorder requiring treatment. A patient in need or an individual in need are used interchangeably herein.
[0106] As used herein, the term "reference" or "reference level" refers to an amount or concentration of the indicated biomarker (e.g., SLFN12, PDE3A, PDE3B, AIP, TRRAP), which may be of interest for comparative purposes. For example, a reference level may be the level of the indicated biomarker expressed as an average of the level of the biomarker from samples taken from a control population of healthy subjects. In some embodiments, a reference level may be the level of the indicated biomarker expressed as an average of the level of the biomarker measured from a plurality cancer cell lines (e.g., the cancer cell lines measured in FIG. 5). In one embodiment, the "reference" is a standard or control condition. Suitable samples or references for determining reference levels include healthy cells, cells nonresponsive to chemically induced complex formation (e.g., cells nonresponsive to PDE3A modulation, cells nonresponsive to PDE3B modulation), non-cancerous cells, normal tissue, and the like. In various implementations, the reference may be the average expression level of cancerous cells. In some embodiments, the reference to determine the reference level of an indicated biomarker may be a derived from the subject, a subject known not to suffer from cancer, such as a healthy subject, or a population of subjects. Suitable references include a concurrently run control, or a standard which may be created by assaying one or more non-cancer cells and collecting biomarker data. Thus, the control sample may optionally be a standard that is created and used continuously. The standard includes, for example, the average level of a biomarker in a sample from a non-cancer control group. In certain embodiments, the reference is derived from a sample of a subject known not to suffer from cancer.
[0107] As used herein, and as well understood in the art, "to treat" a condition or "treatment" of the condition (e.g., the conditions described herein, such as cancer) is an approach for obtaining beneficial or desired results, such as clinical results. Treatment of a subject may include a decrease in the proliferation of cancer cells in the subject. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable. "Palliating" a disease, disorder, or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment.
[0108] Chemical Inducers of PDE3A/B-SLFN12 Complexes
[0109] Without wishing to be bound by theory, it is believed that certain chemical agents are able to induce complexation in certain responsive cells between specific phosphodiesterase and schlafen proteins in the presence of AIP and TRRAP. As shown in WO 2017/027854, hereby incorporated by reference in its entirety, increased expression of PDE3A and SLFN12 has been shown to correlate with cytotoxicity of certain chemical agents (i.e., "complex inducers" or "complex inducing active compounds"), such as PDE3A modulators. As shown herein, selective apoptosis of only malignant biomarker positive cells may occur following chemical induction of PDE3A/B-SLFN12 formation. The present disclosure is partially premised on the discovery that AIP and TRRAP peptides are implicated in the apoptotic response of cancer cells. Expression of combinations of these biomarkers have been shown to correlate with complex inducing active compound (e.g., PDE3 modulator, PDE3A modulator, PDE3B modulator) sensitivity. Using AIP and TRRAP as biomarkers allows for further stratification of cells responsive to PDE3A-SLFN12 or PDE3B-SLFN12 complex formation, and particularly, chemically induced complexation. This increased stratification allows for more efficient identification of specific chemical agents for the treatment or prophylaxis of diseases, disorders, or conditions in that patient population with responsive cells. Furthermore, it facilitates identification of patients who will benefit from a treatment with PDE3 modulators, such as a PDE3A modulator and/or a PDE3B modulator operative via PDE3A-SLFN12 and/or PDE3B-SLFN12 complex formation.
[0110] Once a cell is identified as responsive to complex formation by the methods described herein (e.g., by expression of AIP and/or TRAPP; and increased expression of PDE3A or PDE3B relative to a reference, such as a healthy control cell or the average expression level of cancerous cells such as those cells measured in FIG. 5; and increased expression of SLFN12 relative to reference, such as a healthy control cell or the average expression level of cancerous cells), apoptosis of that cell may occur by chemical induction of complex formation by one or more PDE3 modulators Complex inducing active compounds may be delivered to responsive cell lines to induce complexation between PDE3A-SLFN12 and/or PDE3B-SLFN12 which may result in apoptosis in only those cells susceptible to complex formation (e.g., cancer cells). In some embodiments, the PDE3A-SLFN12 complex inducing active compound is a PDE3 modulator, such as a PDE3A modulator or a PDE3B modulator. In various implementations, the PDE3B-SLFN12 complex inducing active compound is a PDE3 modulator, such as a PDE3A modulator or a PDE3B modulator. For example, the complex inducing active compound may be DNMDP or a compound of WO2019/025562.
[0111] The identification of complex inducing active compounds including PDE3 (e.g., PDE3A, PDE3B) modulators suitable for use as therapeutic agents to induce PDE3A/B-SLFN12 complex formation may be made with a phenotypic screen designed to identify cytotoxic small molecules that preferentially kill cancer cells over a healthy cell through complex formation of PDE3A-SLFN12 or PDE3B-SLFN12 upon administration. A predictive chemogenomics approach may complement target-driven drug development programs, which typically consist of extensive in vitro and in vivo target validation. Many U.S. Food and Drug Administration (FDA)-approved targeted therapies have been developed this way, among them small-molecule kinase inhibitors that target oncogenic somatic driver mutations. However, the discovery and development of targeted therapies is often hampered by limitations in knowledge of the biological function of the target, its mechanism of action, and the available chemical matter to selectively modulate the target. The present disclosure is related to increasing that knowledge base.
[0112] Phenotypic screening can discover novel targets for cancer therapy whose specific molecular mechanism is often elucidated by future studies. For example, a phenotypic screen developed to identify small molecules causing synthetic lethality in tp53 mutant cancer cells enabled the serendipitous discovery of a class of cancer-selective cytotoxic agents which act as modulators of phosphodiesterase 3A (PDE3A). Many PDE3A modulators also directly bind PDE3B proteins and a PDE3A modulator may be used to induce complexation between SLFN12 and PDE3B. Cyclic nucleotide phosphodiesterases catalyze the hydrolysis of second messenger molecules cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), and are important in many physiological processes.
[0113] The present disclosure provides methods for identifying subjects that have a malignancy that is likely to respond to PDE3 modulator treatment based on the expression of AIP and/or TRRAP in a subject biological sample comprising a cancer cell which also expresses increased levels PDE3A and SLFN12 or PDE3B and SLFN12 relative to a reference.
[0114] Examples of PDE3A modulators include DNMDP (6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-o- ne)
##STR00001##
and pharmaceutically acceptable salts thereof or a compound of WO2019/025562 such as (6S)-5-[4'-fluoro-2-(trifluoromethyl)biphenyl-4-yl]-6-methyl-3,6-dihydro-- 2H-1,3,4-oxadiazin-2-one (Compound X), 5-{4-[1-(difluoromethyl)-1H-pyrazol-4-yl]-3-(trifluoromethyl)phenyl}-3,6-- dihydro-2H-1,3,4-oxadiazin-2-one, (6S)-5-[4-(2-aminopyridin-4-yl)-3-(trifluoromethyl) phenyl]-6-methyl-3,6-dihydro-2H-1,3,4-oxadiazin-2-one and (6S)-6-methyl-5-{3-(trifluoromethyl)-4-[3-(trifluoromethyl)-1H-pyrazol-1-- yl]phenyl}-3,6-dihydro-2H-1,3,4-oxadiazin-2-one or a pharmaceutically acceptable salt thereof.
[0115] It will be understood that the modulators described above are known in the art. The structures are provided for illustrative purposes. Any discrepancy between the structure and the known drug will be resolved in favor of the known drug. The PDE3 modulator may be in the form of a pharmaceutically acceptable salt.
[0116] It is possible for the PDE3 modulators to have systemic and/or local activity. For this purpose, they can be administered in a suitable manner, such as, for example, via the oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, vaginal, dermal, transdermal, conjunctival, otic route or as an implant or stent. For these administration routes, it is possible for the compounds according to the disclosure to be administered in suitable administration forms.
[0117] For oral administration, it is possible to formulate the compounds according to the disclosure to dosage forms known in the art that deliver the compounds of the disclosure rapidly and/or in a modified manner, such as, for example, tablets (uncoated or coated tablets, for example with enteric or controlled release coatings that dissolve with a delay or are insoluble), orally-disintegrating tablets, films/wafers, films/lyophylisates, capsules (for example hard or soft gelatine capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions. It is possible to incorporate the compounds according to the disclosure in crystalline and/or amorphised and/or dissolved form into the dosage forms.
[0118] Parenteral administration can be effected with avoidance of an absorption step (for example intravenous, intraarterial, intracardial, intraspinal or intralumbal) or with inclusion of absorption (for example intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal). Administration forms which are suitable for parenteral administration are, inter alia, preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophylisates or sterile powders.
[0119] Examples which are suitable for other administration routes are pharmaceutical forms for inhalation (e.g., powder inhalers, nebulizers), nasal drops, nasal solutions, nasal sprays; tablets/films/wafers/capsules for lingual, sublingual or buccal administration; suppositories; eye drops, eye ointments, eye baths, ocular inserts, ear drops, ear sprays, ear powders, ear-rinses, ear tampons; vaginal capsules, aqueous suspensions (lotions, mixturae agitandae), lipophilic suspensions, emulsions, ointments, creams, transdermal therapeutic systems (such as, for example, patches), milk, pastes, foams, dusting powders, implants or stents.
[0120] Diagnostics
[0121] The present disclosure features diagnostic assays for the characterization of cancer. Levels of AIP and/or TRRAP, particularly in connection with levels of PDE3A and/or PDE3B, and levels SLFN12 may be measured in a subject sample and used as an indicator of cancer that is responsive to treatment with a PDE3 modulator. Levels of AIP, TRRAP, PDE3A, PDE3B, or SLFN12 polynucleotides may be measured by standard methods, such as quantitative PCR, Northern Blot, microarray, mass spectrometry, and in situ hybridization. Standard methods may be used to measure levels of AIP, TRRAP, PDE3A, PDE3B, or SLFN12 polypeptides in a biological sample derived from a tumor. Such methods include immunoassay, ELISA, western blotting using an antibody that binds AIP, TRRAP, PDE3A, PDE3B, or SLFN12, and radioimmunoassay. Elevated levels of PDE3A, SLFN12, AIP and/or TRRAP; or PDE3B, SLFN12, AIP and/or TRRAP polynucleotides or polypeptides are considered a positive indicator of a disease, disorder, or condition (e.g., cancer) that is responsive to treatment with a PDE3 modulator.
[0122] Types of Biological Samples
[0123] In characterizing the responsiveness of a malignancy in a subject to modulation to induce complex formation treatment, the level of AIP, TRRAP, PDE3A, PDE3B, and/or SLFN12 expression is measured in different types of biologic samples. In one embodiment, the biologic sample is a tumor sample.
[0124] In most embodiments, PDE3A and SLFN12 or PDE3B and SLFN12 expression is higher in a sample obtained from a subject that is responsive to PDE3 modulator treatment than the level of expression in a non-responsive subject. In certain implementations, PDE3A, PDE3B, and SLFN12 expression is independently at least about 2, 5, 10, 20, or 30-fold higher in a subject with a malignancy than in a reference condition (e.g., a healthy control). In some embodiments, fold change is determined by calculating the difference in expression of the biomarker (e.g., AIP, TRRAP, PDE3A, PDE3B, SLFN12) in a cancer cell vs the level present in a non-responsive cancer cell or the level present in a corresponding healthy control cell. Additionally, the present disclosure is partially premised on the discovery that PDE3A-SLFN12 or PDE3B-SLFN12 complex formation (and thus apoptosis of cells) occurs when the cells also express AlP. It has also been discovered that TRRAP is required for sensitivity to DNMDP. Accordingly, in addition to increased PDE3A and SLFN12 biomarkers, the cells responsive to complex formation may express no alteration or loss, minimal alteration or loss, or increased expression of AIP and/or TRRAP expression as compared to a reference. For example, the responsive cells may have more than 50% or more than 60% or more than 70% or more than 80% or more than 90% or more than 100% expression of AIP and/or TRRAP as compared to a reference. In certain embodiments, the cell may be considered to not express AIP and/or TRRAP if the number of copies of the biomarker per cellular genome is less than 1 or less than 2.sup.-1 or less than or less than 2.sup.-2 or or less than 2.sup.-3 or less than 2.sup.-4 or less than 2.sup.-5. Conversely, the cell may be considered to express AIP and/or TRRAP if the number of copies of the biomarker per cellular genome is greater than 1 or greater than 2.sup.-1 or greater than or greater than 2.sup.-2 or greater than 2.sup.-3 or greater than 2.sup.-4 or greater than 2.sup.-5. In certain embodiments, the reference is the average expression level of an indicated biomarker (e.g., PDE3A, PDE3B, SLFN12, AIP, TRRAP) in all cell lines for which data is shown in FIG. 5. In various implementations AIP and/or TRAPP is considered to be expressed in a cell if the expression is greater than the the average expression level of the biomarker in all cell lines for which data is shown in FIG. 5 (i.e., the reference for AIP and TRRAP may be the average expression level of the biomarker in all cell lines for which data is shown in FIG. 5). In some embodiments, PDE3A, PDE3B, and/or SLFN12 is considered to have increased expression in a cell if the expression is greater than a healthy control cell (i.e. the reference for PDE3A, PDE3B, and/or SLFN12 may be a healthy control cell). In some embodiments, PDE3A, PDE3B, and/or SLFN12 is considered to have increased expression in a cell if the expression is greater than the average expression levels of the cancer cells in FIG. 5.
[0125] Selection of a Treatment Method
[0126] As reported herein, subjects suffering from a malignancy may be tested for AIP and/or TRRAP expression in the course of selecting a treatment method or during the treatment method. In some embodiments, patients characterized as having:
[0127] (i) AIP and/or TRRAP expression (e.g. as determined by the average expression level in cancer cells such as that shown in FIG. 5),
[0128] (ii) increased expression of PDE3A or PDE3B relative to a reference (e.g., a healthy cell, a value determined from the average expression level from a healthy sample population, as determined by the average expression level in cancer cells such as that shown in FIG. 5), and
[0129] (iii) increased expression of SLFN12 relative to a reference (e.g., a healthy cell, a value determined from the average expression level from a healthy sample population, a value determined from the average expression level in cancer cells for example as determined from the cells measured in FIG. 5);
are identified as responsive to complex formation and PDE3 modulator treatment. For example, those patients characterized as having:
[0130] (i) AIP and TRRAP expression (e.g. as determined by the average expression level in cancer cells such as that shown in FIG. 5),
[0131] (ii) increased expression of PDE3A or PDE3B relative to a reference (e.g., a healthy cell, a value determined from the average expression level from a healthy sample population, a value determined from the average expression level in cancer cells for example as determined from the cells measured in FIG. 5), and
[0132] (iii) increased expression of SLFN12 relative to a reference (e.g., a healthy cell, a value determined from the average expression level from a healthy sample population, a value determined from the average expression level in cancer cells for example as determined from the cells measured in FIG. 5);
may be identified as responsive to complex formation and PDE3 modulator treatment. In certain implementations, patients characterized as having:
[0133] (i) AIP and/or TRRAP expression (e.g. as determined by the average expression level in cancer cells such as that shown in FIG. 5),
[0134] (ii) increased expression of PDE3A relative to a reference (e.g., a healthy cell, a value determined from the average expression level from a healthy sample population, a value determined from the average expression level in cancer cells for example as determined from the cells measured in FIG. 5), and
[0135] (iii) increased expression of SLFN12 relative to a reference (e.g., a healthy cell, a value determined from the average expression level from a healthy sample population, a value determined from the average expression level in cancer cells for example as determined from the cells measured in FIG. 5);
are identified as responsive to complex formation and PDE3 modulator (e.g., PDE3A modulator, PDE3B modulator) treatment. Those patients characterized as having:
[0136] (i) AIP and TRRAP expression (e.g. as determined by the average expression level in cancer cells such as that shown in FIG. 5),
[0137] (ii) increased expression of PDE3A relative to a reference (e.g., a value determined from the average expression level from a healthy sample population, a value determined from the average expression level in cancer cells for example as determined from the cells measured in FIG. 5), and
[0138] (iii) increased expression of SLFN12 relative to a reference;
may be identified as responsive to complex formation and PDE3 modulator (e.g., PDE3A modulator, PDE3B modulator) treatment. In various implementations, patients characterized as having:
[0139] (i) AIP and/or TRRAP expression,
[0140] (ii) increased expression of PDE3B relative to a reference, and
[0141] (iii) increased expression of SLFN12 relative to a reference;
are identified as responsive to complex formation and PDE3 modulator (e.g., PDE3A modulator, PDE3B modulator) treatment. In some embodiments, patients characterized as having:
[0142] (i) AIP and TRRAP expression (e.g. as determined by the average expression level in cancer cells such as that shown in FIG. 5),
[0143] (ii) increased expression of PDE3B relative to a reference (e.g., a value determined from the average expression level from a healthy sample population, as determined by the average expression level in cancer cells such as that shown in FIG. 5), and
[0144] (iii) increased expression of SLFN12 relative to a reference (e.g., a value determined from the average expression level from a healthy sample population, as determined by the average expression level in cancer cells such as that shown in FIG. 5);
are identified as responsive to complex formation and PDE3 modulator (e.g., PDE3A modulator, PDE3B modulator) treatment.
[0145] In certain embodiments for the selection of treatment methods described above the reference is the average expression level of all cell lines for which data is shown in FIG. 5.
[0146] In certain embodiments, the disclosure provides a method for identifying a subject having cancer responsive to treatment with a PDE3 modulator, particularly a compound of WO2019/025562, hereby incorporated by reference in its entirety and specifically in relation to compounds of formula (I) such as those on page 49, line 35-page 75, line 11. In various implementations, the PDE3 modulator may be selected from (6S)-5-[4'-fluoro-2-(trifluoromethyl)biphenyl-4-yl]-6-methyl-3,6-dihydro-- 2H-1,3,4-oxadiazin-2-one, 5-{4-[1-(difluoromethyl)-1H-pyrazol-4-yl]-3-(trifluoromethyl)phenyl}-3,6-- dihydro-2H-1,3,4-oxadiazin-2-one, (6S)-5-[4-(2-aminopyridin-4-yl)-3-(trifluoromethyl) phenyl]-6-methyl-3,6-dihydro-2H-1,3,4-oxadiazin-2-one and (6S)-6-methyl-5-{3-(trifluoromethyl)-4-[3-(trifluoromethyl)-1H-pyrazol-1-- yl]phenyl}-3,6-dihydro-2H-1,3,4-oxadiazin-2-one or a salt thereof, the method comprising determining
[0147] a) AIP and TRRAP expression,
[0148] b) increased expression of PDE3A or PDE3B relative to a reference (e.g., the level present in a corresponding healthy/control cell or the average expression level of cancer cell lines such as those illustrated in FIG. 5), and
[0149] c) increased expression of SLFN12 relative to a reference (e.g., the level present in a corresponding healthy/control cell or the average expression level of cancer cell lines such as those illustrated in FIG. 5);
[0150] in a sample; thereby identifying the subject as having a cancer responsive to treatment with a PDE3 modulator.
[0151] In certain embodiments, the method above can be used to identify a subject as having a cancer that is less likely to respond to treatment comprising a PDE3 modulator mentioned herein the method comprising:
[0152] a) determining AIP and/or TRRAP expression, PDE3a or PDE3B expression and SLFN12 expression in a sample from said subject and
[0153] b) identifying the subject as being less likely to respond to treatment comprising a PDE3 modulator when AIP and/or TRRAP are absent.
[0154] The present disclosure also relates to the use of AlP for stratifying in vitro a cancer patient or a sample from a cancer patient disposed to respond treatment with a PDE3 modulator mentioned herein.
[0155] The use of a capture reagent, such as an antibody, that binds to or interacts with AlP for stratifying in vitro a cancer patient or sample from a cancer patient disposed to respond to a PDE3 modulator treatment mentioned herein is contemplated within the present disclosure.
[0156] The use of a capture reagent that binds to or interacts with TRRAP for stratifying in vitro a cancer patient or sample from a cancer patient disposed to respond to a PDE3 modulator treatment mentioned herein is contemplated within the present disclosure as well.
[0157] The use of a capture reagent that binds to or interacts with SLFN12 for stratifying in vitro a cancer patient or sample from a cancer patient disposed to respond to a PDE3 modulator treatment mentioned herein is contemplated within the present disclosure as well.
[0158] The use of a PDE3 modulator mentioned herein for the treatment of cancer in a subject characterized by the expression of AIP, TRRAP and increased expression of PDE3A or PDE3B and SLFN12 is contemplated within the present disclosure as well.
[0159] The cells identified as being responsive to complex formation (e.g., as chemically induced by PDE3 modulation) may be hyperproliferative cells related to a hyperproliferative disease, disorder, or condition. Such identification may be used in the treatment and/or prevention of various hyperproliferative diseases, disorders, or conditions, such as a myeloproliferative disorder or cancer. In specific embodiments, the cell may be a cancer cell. In some implementations, the may be a cancer cell selected from bladder-, brain-, breast-, cervical-, colorectal-, endometrial-, esophageal-, gallbladder-, gastric-, glioblastoma-, kidney-, leukemia- (e.g., acute myelogenous leukemia-, chronic myelogenous leukemia-, chronic lymphocytic leukemia-), liver- (e.g., hepatocellular carcinoma-, intrahepatic cholangiocarcinoma-, angiosarcoma-, hemangiosarcoma-, hepatoblastoma-), lung- (e.g., non-small cell lung cancer-, small cell lung cancer-, mesothelioma-), melanoma-, ovarian-, pancreatic-, prostate-, multiple myeloma-, sarcoma- (e.g., osteosarcoma-, soft-tissue sacrcoma-), thyroid-, urinary tract-, uterine cancer cells. In certain implementations the cell may be a hematopoietic cancer cell, such as acute lymphoblastic leukemia-, acute myelogenous leukemia-, chronic lymphocytic leukemia-, chronic myelogenous leukemia-, acute monocytic leukemia-, Hodgkin's lymphoma-, or non-Hodgkin's lymphoma cells. Other hyperproliferative disease, disorder, or conditions considered within the scope of the disclosure include myeloproliferative diseases, such as essential thromobocytosis.
[0160] Kits
[0161] The disclosure provides kits for characterizing the responsiveness of a subject to complex formation and PDE3 modulator treatment.
[0162] In certain embodiments, the kit may include a therapeutic composition containing an effective amount of a PDE3 modulator (e.g., PDE3A modulator, PDE3B modulator) in unit dosage form.
[0163] In certain implementations, a diagnostic kit of the disclosure provides one or more reagents for measuring expression of AIP, TRRAP, PDE3A, PDE3B, SLFN12, and combinations thereof. Such reagents include one or more capture molecules (e.g., antibodies that recognize a polypeptide selected from AIP, TRRAP, PDE3A, PDE3B or SLFN12). In some embodiments, the kit comprises a reagent for measuring the expression of AIP, a reagent for measuring the expression of PDE3A, and a reagent for measuring the expression of SLFN12. In some embodiments, the kit comprises a reagent for measuring the expression AlP, a reagent for measuring the expression of PDE3B, and a reagent for measuring the expression of SLFN12. In some embodiments, the kit comprises a reagent for measuring the expression of TRRAP, a reagent for measuring the expression of PDE3A, and a reagent for measuring the expression of SLFN12. In some embodiments, the kit comprises a reagent for measuring the expression TRRAP, a reagent for measuring the expression of PDE3B, and a reagent for measuring the expression of SLFN12. In some embodiments, the kit comprises a reagent for measuring the expression of AIP, a reagent for measuring the expression of TRRAP, a reagent for measuring the expression of PDE3A, and a reagent for measuring the expression of SLFN12. In some embodiments, the kit comprises a reagent for measuring the expression AIP, a reagent for measuring the expression of TRRAP, a reagent for measuring the expression of PDE3B, and a reagent for measuring the expression of SLFN12. In some embodiments, the kit comprises a reagent for measuring the expression AIP, a reagent for measuring the expression of TRRAP, a reagent for measuring the expression of PDE3A, a reagent for measuring the expression of PDE3B, and a reagent for measuring the expression of SLFN12.
[0164] In some embodiments the kit comprises a PDE3 modulator mentioned herein together with reagents for measurement of expression of AIP, TRRAP, PDE3A or PDE3B, and SLFN12.
[0165] The kit may comprise a sterile container which contains a therapeutic or diagnostic composition-such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. In certain implementations, the container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments. If desired, the kit further comprises instructions for measuring biomarker (e.g., PDE3A, PDE3B, SLFN12, TRRAP, AIP) expression and/or instructions for administering the PDE3 modulator to a subject having a malignancy, e.g., a malignancy selected as responsive to PDE3A modulator treatment. In particular embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of malignancy or symptoms thereof, precautions; warnings; indications; counter-indications; over dosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
[0166] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. These techniques are applicable to the production of the polynucleotides and polypeptides of the disclosure, and, as such, may be considered in making and practicing the disclosure. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
[0167] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure.
EXAMPLES
[0168] The following examples illustrate specific aspects of the instant description. The examples should not be construed as limiting, as the example merely provides specific understanding and practice of the embodiments and its various aspects.
Example 1: Profiling for Sensitivity to PDE3A Modulation
[0169] To measure cancer cell death in response to 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-on- e (DN/DP) treatment, cells were plated in 384w assay plates at the following cell density per well: 500 cells of HeLa (DMEM), A2058 (DMEM), HMCB (EMEM), IGR37 (DMEM), NCIH1734 (RPMI), 750 cells of CAL51 (DMEM), COL0741 (RPMI), DKMG (RPMI), GB1 (EMEM), HEL (RPMI), HEL9217 (RPMI), JHUEM1 (DMEM+F12), L3.3 (RPMI) and TE4 (RPMI), HCC15 (RPMI), UACC257 (RPMI), 1000 cells for HUT78 (IMEM), NCIH1563 (RPMI), NCIH2122 (RPMI), NCIH2172 (RPMI), RVH421 (RPMI) and SKMEL3 (McCoy's 5A), 1500 cells for C32 (EMEM), HS578T (DMEM) and JHOM1 (DMEM+F12). Cells were incubated at 37.degree. C. overnight and then treated with a DNMDP dose dilution series using an HP D300 digital dispenser. After 72 hours, the viability of cells in each well were measured by Cell Titer Glo (Promega G755B and G756B). Percent viability values were determined using the values from untreated wells and AUC values were calculated using a 4-parameter fit. DNMDP was purchased from Life Chemicals (F1638-0042) and trequinsin was purchased from Sigma-Aldrich (T2057).
[0170] 1400 cells per well were seeded in a 96 well plate in media that had been centrifuged at 500.times.g for 5 min to remove particulates. The next day, the red fluorescent DNA-staining dye, Incucyte Nuclite Rapid Red, and green fluorescent apoptosis dye, Incucyte Caspase-3/7 Green Apoptosis Reagent (Essen Biosciences), were added in 2 .mu.l FBS to a final concentration of 1:1000 and 1:1500, respectively. Two hours later, [2 .mu.M DNMDP+0.2% DMSO] or 0.2% DMSO was added. Because even sensitive cells sometimes divided before 24 hours, cells were tracked starting at 24 hours, although cells that apoptosed before 24 hours were also counted. For the washout study, the media was removed from DNMDP treated cells at 72 h, the cells were rinsed with media, and incubation was continued in the absence of DNMDP. Cells were tracked starting at 72 h. Images were taken every 1 h up to 96 h, and every 2 h thereafter, with an Incucyte S3 machine (Essen Biosciences). Three channels were recorded: phase contrast, red fluorescence (DNA), and green fluorescence (apoptosis). For cell tracking, a movie superimposing all three channels was analyzed. To avoid effects due to depletion of media components over time, cells were followed up to the last hour before DMSO control cells started to show slowed division or increased apoptosis (136 h for HeLa, 194 h for SKMEL3, 160 h for GB1, 130 h for TE4, 130 h for A2058, 144 h for DKMG, 106 h for HS578T, 186 h for H2172, 220 h for C32).
[0171] Melanoma cell lines were tested for sensitivity to DNMDP and the biomarkers of these cells were identified. Biomarker expression thresholds were optimized for positive predictive value and sensitivity. Of the 49 melanoma cell lines tested, seven expressed elevated levels of PDE3A and SLFN12 and one expressed elevated levels of the related protein PDE3B. FIG. 1 compares the reads per kilobase of transcript per million mapped reads (RKPM) for PDE3A and SLFN12 across hundreds of cancer cell lines and identifies the biomarker positive cell lines. Table 1 illustrates the biomarker expression and DNMDP response for 7 melanoma cell lines among the biomarker-positive cell lines in FIG. 1. The eighth sensitive melanoma cell line, RVH421, expressed elevated levels of the related protein, PDE3B. In Table 1, the biomarker mRNA levels expressed as log.sub.2(RPKM+1) and the area under the curve (AUC) is calculated on a scale from 0-4. Previous analysis of sensitivity data defined the positive predictive value (PPV) of the combined SLFN12/PDE3A biomarker to be about 50%, but the optimized biomarker thresholds result in a PPV of 62%, with sensitive defined by an AUC of less than 2.8, equivalent to an AUC of 1.6 on a scale of 0-4.
TABLE-US-00012 TABLE 1 PDE3A PDE3B SLFN12 Cell Line Lineage expression expression expression AUC HeLa cervical 5.65 1.68 2.85 0.36 IGR37 melanoma 5.29 1.18 2.50 1.17 COLO741 melanoma 4.30 1.20 2.41 1.19 SKMEL3 melanoma 2.69 1.28 2.96 1.74 HMCB melanoma 3.82 1.72 2.09 1.85 A2058 melanoma 4.64 1.32 2.02 2.11 C32 melanoma 2.97 1.32 3.37 3.13 UACC257 melanoma 4.90 1.95 2.39 4.00 RVH421 melanoma 0.16 2.66 2.16 3.02
[0172] All but one of these eight melanoma cell lines were sensitive to DNMDP. FIG. 2A illustrates the dose response of Cell Titer-Glo assay measurements on these eight cell lines with administration of DNMDP, whereas FIGS. 2B and 2C illustrate the response of all tested PDE3A/SLFN12 biomarker-positive cell lines from multiple disease indications (RVH421 is PDE3B/SLFN12 biomarker-positive). As can be seen in FIG. 2A, C32 and RVH421 are only partially sensitive, and UACC257 cells are completely insensitive. Further experimentation has linked the UACC257 insensitivity to its lack of AIP expression (see, e.g., Example 3). Additionally, it can be seen that sensitivity is not binary; rather dose response curves showed a continuous gradient of inhibition. Based on maximum viability values, the tested cell lines could be split into strongly sensitive cell lines (13 cell lines with <25% maximum viability, FIG. 2B), partially sensitive lines (7 cell lines with 25-75% maximum viability; FIG. 2C), and insensitive cell lines (2 cell; lines with 100% maximal viability; FIG. 2C).
Example 2: CRISPR Knockout
[0173] PDE3A CRISPR KO cells (sgRNA #2) were generated according to de Waal et al, 2016 (2), hereby incorporated by reference in its entirety. CRISPR target sites for PDE3B and AIP were identified using the CHOPCHOP CRISPR Design Tool (chopchop.cbu.uib.no). For cloning of sgRNAs, forward and reverse oligos were annealed, phosphorylated and ligated into a BsmBI-digested lentiCRISPRv2 vector. Lentivirus carrying each guide construct was packaged as described above and used to infect target cells. Transduced target cells were selected using 1 .mu.g/ml puromycin and passaged for 7 days before use.
[0174] FIG. 3A illustrates the survival of cells in the presence of DNMDP that have had CRISPR knockout of SLFN12. FIG. 3B illustrates the survival of cells in the presence of DNMDP that have had CRISPR knockout of PDE3A (with and without ectopic expression of PDE3B). As can be seen, SLFN12 knockout abrogates all measured sensitivity to PDE3 modulators. Moreover, PDE3B can support DNMDP sensitivity in the absence of PDE3A expression, and ectopic expression of PDE3B can furthermore support DNMDP sensitivity in PDE3A knockout HeLa cells. The survival in FIGS. 3A and 3B was measured with a 72 h Cell Titer-Glo assay and CRISPR was performed with sg4, SLNF12 CRISPR guide RNA #4.
[0175] In a genome wide CRISPR screen, genes were identified as important for cancer cell killing in HeLa cells. The Brunello CRISPR library was used for the DNMDP resistance screen. Lentiviral infection was carried out in duplicate and for each replicate with enough HeLa cells to achieve >1000 infected cells per library member (80000 sgRNAs, >8.times.10.sup.7 cells total) and at low multiplicity of infection (MOI) to achieve transduction of a single sgRNA per cell. Infection efficiencies for the two replicates were 24% and 31% respectively, corresponding to a MOI of about 0.3, meaning about 85% of infected cells would be predicted to have single sgRNA integration. At the time of infection, HeLa cells were resuspended in media and mixed with Brunello library virus in the presence of 8 .mu.g/ml polybrene (library lentivirus provided by the Genetic Perturbation Platform at the Broad Institute), plated in 12 well dishes at 3.times.10.sup.6 cells per well, and spun at 931.times.g for 2 h at 30.degree. C. 2 h after the spin infection, virus-containing media was removed and fresh media was added for incubation overnight. The day after the infection, cells were trypsinized and pooled into T225 flasks at 50% confluence (1.6.times.10.sup.7 cells per flask) and puromycin was added to 1 .mu.g/ml to select for infected cells. At the same time, in-line infection efficiency assays were performed by comparing cell counts after puromycin selection to those without selection. After 4 days of puromycin selection, infected cells were collected and passaged in T225 flasks at 25% confluence (8.times.10.sup.6 cells per flask) for three additional days to allow CRISPR KO to complete. Cells were collected at 8 days after infection, and 8.times.10.sup.7 cells each were split into DMSO control arm (plating at 8.times.10.sup.6 cells per T225 flask) or 25 nM DNMDP treatment arm (plating at 2.times.10.sup.7 cells per T225 flask). Cells were passaged every 3 to 4 days at 25% confluence for the next 14 days. For the DMSO arm, 8.times.10.sup.7 cells were maintained at every passage, whereas all surviving cells were passaged for the DNMDP arm. After 14 days of compound treatment, cells were harvested, washed with cold PBS and flash frozen at 2.times.10.sup.7 cells (DMSO arm) or less portions for genomic DNA isolation. Genomic DNA was isolated using the Nucleospin Blood XL kit (DMSO samples, 4 preps to cover 8.times.10.sup.7 cells, Machere-Nagel 740950.50) or the QIAamp DNA Blood Mini kit (DNMDP-treated samples, Qiagen 51104). PCR amplification of sgRNA tags and pooled library sequencing were carried out as described in Sanson, et al. "Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities" Nat Commun 9(1):5416 ("Sanson"), hereby incorporated by reference in its entirety.
[0176] CRISPR screen data analysis was done largely as described in Sanson. Briefly, deconvolution of sequencing reads yielded read counts for each sgRNA under each replicate treatment condition. Log 2-Normalized-Reads for each guide per condition was calculated using the formula log 2 (guide/total*1000000+1) and averaged across the two replicates. Subtracting DMSO values from those for 25 nM DNMDP generated Log-Fold-Change values for each sgRNA, which were then averaged across all sgRNAs targeting the same gene to generate gene-level Average-Log-Fold-Change score. To statistically evaluate gene-level enrichment in DNMDP treatment relative to DMSO, sgRNAs were rank ordered based on Average-Log-Fold-Change, and p-values for each sgRNA relative to the rank order were determined by running a hypergeometric distribution without replacement, equivalent to a one-sided Fisher's exact test. The average of the negative log 10 p values for each sgRNA targeting the same gene was calculated to generate the average negative log 10 p-value for each gene. A volcano plot was generated using the average-log 2-fold-change and the average negative log 10 p-value for all genes with 3 to 8 sgRNAs per gene to visualize gene enrichments after the positive selection of 25 nM DNMDP treatment.
[0177] AIP, SLFN12, and PDE3A knockout cause the greatest increase in cell survival in the presence of 25 nm DN/DP. FIG. 4 illustrates the results of the CRISPR screen comparing the log fold change (LFC) of gene CRISPR guide representation as compared to -log(p-value) of the screen (each as compared to HeLa cells). The gRNA best supporting survival in the presence of DNMDP was specific for the AIP. As expected, SLFN12 and PDE3A knockout also strongly supported cell survival in the presence of DNMDP, ranking second and third behind AlP, respectively. Knockout of the histone acetyltransferase complex protein, transformation/transcription domain associated protein (TRRAP), exhibited as significant but much weaker phenotype as well.
[0178] The CRISPR screen allowed for identification of the aryl hydrocarbon receptor interacting protein as having potent effect on cell survival rates. AIP is a co-chaperone protein that regulates stability and subcellular localization of the aryl hydrocarbon receptor and other proteins, as described in Trivellin, G. and M. Korbonits, J Endocrinol 210 (2011): 137-55, hereby incorporated by reference in its entirety. Of the cancer cell lines tested, only a single cancer cell line, UACC257, the biomarker-positive but DNMDP-resistant melanoma cell line lacked AIP expression. This can be seen in FIG. 5 where the x axis is plotted by log.sub.2 copy number, and the y axis by gene expression. As can be seen, UACC257 does not express AIP while the remaining cell lines do.
[0179] Furthermore, that AIP knockout eliminates HeLa cell response to DNMDP was validated with independent gRNAs as shown in FIG. 6A. Similar results were observed upon knockout of AIP from the melanoma cell line A2058 (FIG. 6B). Additionally, AIP knockout in these cell lines resulted in decreased PDE3A protein expression (FIG. 6C).
Example 3: AIP Knockout and Complex Formation
[0180] Because decreased PDE3A protein expression could impact DNMDP-induced PDE3A-SLFN12 complex formation, the effects of AIP knockout on this complex formation were also measured. As there is no good antibody for SLFN12 protein, we ectopically expressed V5-tagged SLFN12 in parental or AIP-knockout HeLa cells, immunoprecipitated endogenous PDE3A, and assessed whether V5-SLFN12 could be detected in the immunoprecipatites. Cells were plated in 10 cm petri dishes and collected at 50-90% confluence. For PDE3A immunoblotting in biomarker positive cells and in AIP KO cells, cells were seeded in 15 cm plates at similar density as in viability assays with a vessel scaling factor of 5000, e.g., 500 cells per well was scaled to 10.sup.6 cells per 10 cm plate or 2.5.times.10.sup.6 cells per 15 cm plate, and then cultured for 72 hours before collection. Cell pellets were lysed at 4.degree. C. for 20 minutes in modified RIPA buffer (150 mM NaCl, 10% glycerol, 50 mM Tris-Cl pH 8.0, 50 mM MgCl2, 1% NP-40) supplemented with EDTA-free protease inhibitors (Sigma-Aldrich 4693159001) and PhosSTOP phosphatase inhibitors (Sigma-Aldrich 4906837001). Lysates were clarified by centrifugation at 13,000 rpm.times.10 min at 4.degree. C. and quantified using BCA protein assays (Thermo Fisher Scientific 23225). Clarified lysates were resolved on 4-12% Bis-Tris PAGE gels, transferred to nitrocellulose membranes (Thermo Fisher Scientific IB23001) and immunoblotted with primary antibodies against PDE3A (Bethyl 302-740A, 1:2000), V5 (Life Technologies R96205 at 1:5,000), AIP (Thermo Fisher Scientific MA3-16515 at 1:2000), Vinculin (Sigma-Aldrich V9264 at 1:5,000), GAPDH (Cell Signaling Technology 2118 at 1:2000) and secondary antibodies from LiCOR Biosciences (92632210 and 926068021, each at 1:10,000). Blots were washed and imaged using a LiCOR Odyssey infrared imager, and fluorescent signals quantified using the Image Studio software provided by the LiCOR manufacturer.
[0181] Genomic DNA was isolated from cells using QIAamp DNA mini kit (Qiagen 51304) and SLFN12 genomic region was amplified by PCR using Q5 High-Fidelity 2.times. Master Mix (New England Biolabs M0492) and primers SLFN12_2_F or SLFN12_428_F and SLFN12_858_R. PCR products were purified using QIAquick PCR Purification Kit (Qiagen 28104) and send for sequencing using Forward or Reverse primers used for PCR. Sequencing reads were aligned to reference sequence using Benchling alignment tools.
[0182] AIP knockout completely abolished PDE3A-SLFN12 complex formation in response to DNMDP (FIG. 7), confirming that AIP functions upstream of DNMDP-induced complex formation. The partial decrease in PDE3A protein levels shown in FIG. 6C cannot fully account for the effects of AIP knockout on PDE3A-SLFN12 complex formation. For example, A549 cells, which express sufficient endogenous PDE3A to support partial sensitivity to complex inducing active compounds upon ectopic expression of SLFN12, express similar levels or even less PDE3A than these AIP knockout cells.
[0183] AIP was required for cancer cell killing in response to Compound X (also termed (6S)-5-[4'-fluoro-2-(trifluoromethyl)biphenyl-4-yl]-6-methyl-3, 6-dihydro-2H-1,3,4-oxadiazin-2-one) (FIG. 8A). Cervical cancer cell viability was reduced at increasing concentrations of Compound X (FIG. 8A). Cancer cell killing required AIP, as no cancer cell killing was observed in HeLa cells lacking AIP, i.e., HeLa cells having an AIP knock out. AlP knockout also abolished PDE3A-SLFN12 complex formation in response to treatment with Compound X as shown at FIG. 8B. In FIG. 8B an anti-PDE3A antibody was used to pull down a PDE3A-SLFN12 complex. Complex formation between PDE3A and FLAG-tagged SLFN12 was induced in the presence of Compound X and in the presence of DNMDP. Complex formation was not observed when AIP was knocked out using Crispr (KO sg2, KO sg3). HeLa cells were treated with 10 M DNMDP or 10 .mu.M Compound X. These results indicate that AIP can act as a marker for responsiveness to Compound X.
[0184] As various changes can be made in the above-described subject matter without departing from the scope and spirit of the present disclosure, it is intended that all subject matter contained in the above description, or defined in the appended claims, be interpreted as descriptive and illustrative of the present disclosure. Many modifications and variations of the present disclosure are possible in light of the above teachings. Accordingly, the present description is intended to embrace all such alternatives, modifications and variances which fall within the scope of the appended claims.
Sequence CWU
1
1
1117319DNAHomo sapiens 1gggggccact gggaattcag tgaagagggc accctatacc
atggcagtgc ccggcgacgc 60tgcacgagtc agggacaagc ccgtccacag tggggtgagt
caagccccca cggcgggccg 120ggactgccac catcgtgcgg accccgcatc gccgcgggac
tcgggctgcc gtggctgctg 180gggagacctg gtgctgcagc cgctccggag ctctcggaaa
ctttcctccg cgctgtgcgc 240gggctccctg tcctttctgc tggcgctgct ggtgaggctg
gtccgcgggg aggtcggctg 300tgacctggag cagtgtaagg aggcggcggc ggcggaggag
gaggaagcag ccccgggagc 360agaagggggc gtcttcccgg ggcctcgggg aggtgctccc
gggggcggtg cgcggctcag 420cccctggctg cagccctcgg cgctgctctt cagtctcctg
tgtgccttct tctggatggg 480cttgtacctc ctgcgcgccg gggtgcgcct gcctctggct
gtcgcgctgc tggccgcctg 540ctgcgggggg gaagcgctcg tccagattgg gctgggcgtc
ggggaggatc acttactctc 600actccccgcc gcgggggtgg tgctcagctg cttggccgcc
gcgacatggc tggtgctgag 660gctgaggctg ggcgtcctca tgatcgcctt gactagcgcg
gtcaggaccg tgtccctcat 720ttccttagag aggttcaagg tcgcctggag accttacctg
gcgtacctgg ccggcgtgct 780ggggatcctc ttggccaggt acgtggaaca aatcttgccg
cagtccgcgg aggcggctcc 840aagggagcat ttggggtccc agctgattgc tgggaccaag
gaagatatcc cggtgtttaa 900gaggaggagg cggtccagct ccgtcgtgtc cgccgagatg
tccggctgca gcagcaagtc 960ccatcggagg acctccctgc cctgtatacc gagggaacag
ctcatggggc attcagaatg 1020ggaccacaaa cgagggccaa gaggatcaca gtcttcagga
accagtatta ctgtggacat 1080cgccgtcatg ggcgaggccc acggcctcat taccgacctc
ctggcagacc cttctcttcc 1140accaaacgtg tgcacatcct tgagagccgt gagcaacttg
ctcagcacac agctcacctt 1200ccaggccatt cacaagccca gagtgaatcc cgtcacttcg
ctcagtgaaa actatacctg 1260ttctgactct gaagagagct ctgaaaaaga caagcttgct
attccaaagc gcctgagaag 1320gagtttgcct cctggcttgt tgagacgagt ttcttccact
tggaccacca ccacctcggc 1380cacaggtcta cccaccttgg agcctgcacc agtacggaga
gaccgcagca ccagcatcaa 1440actgcaggaa gcaccttcat ccagtcctga ttcttggaat
aatccagtga tgatgaccct 1500caccaaaagc agatccttta cttcatccta tgctatttct
gcagctaacc atgtaaaggc 1560taaaaagcaa agtcgaccag gtgccctcgc taaaatttca
cctctttcat cgccctgctc 1620ctcacctctc caagggactc ctgccagcag cctggtcagc
aaaatttctg cagtgcagtt 1680tccagaatct gctgacacaa ctgccaaaca aagcctaggt
tctcacaggg ccttaactta 1740cactcagagt gccccagacc tatcccctca aatcctgact
ccacctgtta tatgtagcag 1800ctgtggcaga ccatattccc aagggaatcc tgctgatgag
cccctggaga gaagtggggt 1860agccactcgg acaccaagta gaacagatga cactgctcaa
gttacctctg attatgaaac 1920caataacaac agtgacagca gtgacattgt acagaatgaa
gatgaaacag agtgcctgag 1980agagcctctg aggaaagcat cggcttgcag cacctatgct
cctgagacca tgatgtttct 2040ggacaaacca attcttgctc ccgaacctct tgtcatggat
aacctggact caattatgga 2100gcagctaaat acttggaatt ttccaatttt tgatttagtg
gaaaatatag gaagaaaatg 2160tggccgtatt cttagtcagg tatcttacag actttttgaa
gacatgggcc tctttgaagc 2220ttttaaaatt ccaattaggg aatttatgaa ttattttcat
gctttggaga ttggatatag 2280ggatattcct tatcataaca gaatccatgc cactgatgtt
ttacatgctg tttggtatct 2340tactacacag cctattccag gcctctcaac tgtgattaat
gatcatggtt caaccagtga 2400ttcagattct gacagtggat ttacacatgg acatatggga
tatgtattct caaaaacgta 2460taatgtgaca gatgataaat acggatgtct gtctgggaat
atccctgcct tggagttgat 2520ggcgctgtat gtggctgcag ccatgcacga ttatgatcat
ccaggaagga ctaatgcttt 2580cctggttgca actagtgctc ctcaggcggt gctatataac
gatcgttcag ttttggagaa 2640tcatcacgca gctgctgcat ggaatctttt catgtcccgg
ccagagtata acttcttaat 2700taaccttgac catgtggaat ttaagcattt ccgtttcctt
gtcattgaag caattttggc 2760cactgacctg aagaaacact ttgacttcgt agccaaattt
aatggcaagg taaatgatga 2820tgttggaata gattggacca atgaaaatga tcgtctactg
gtttgtcaaa tgtgtataaa 2880gttggctgat atcaatggtc cagctaaatg taaagaactc
catcttcagt ggacagatgg 2940tattgtcaat gaattttatg aacagggtga tgaagaggcc
agccttggat tacccataag 3000ccccttcatg gatcgttctg ctcctcagct ggccaacctt
caggaatcct tcatctctca 3060cattgtgggg cctctgtgca actcctatga ttcagcagga
ctaatgcctg gaaaatgggt 3120ggaagacagc gatgagtcag gagatactga tgacccagaa
gaagaggagg aagaagcacc 3180agcaccaaat gaagaggaaa cctgtgaaaa taatgaatct
ccaaaaaaga agactttcaa 3240aaggagaaaa atctactgcc aaataactca gcacctctta
cagaaccaca agatgtggaa 3300gaaagtcatt gaagaggagc aacggttggc aggcatagaa
aatcaatccc tggaccagac 3360ccctcagtcg cactcttcag aacagatcca ggctatcaag
gaagaagaag aagagaaagg 3420gaaaccaaga ggcgaggaga taccaaccca aaagccagac
cagtgacaat ggatagaatg 3480ggctgtgttt ccaaacagat tgacttgtca aagactctct
tcaagccagc acaacattta 3540gacacaacac tgtagaaatt tgagatgggc aaatggctat
tgcattttgg gattcttcgc 3600attttgtgtg tatattttta cagtgaggta cattgttaaa
aactttttgc tcaaagaagc 3660tttcacattg caacaccagc ttctaaggat tttttaagga
gggaatatat atgtgtgtgt 3720gtatataagc tcccacatag atacatgtaa aacatattca
cacccatgca cgcacacaca 3780tacacactga aggccacgat tgctggctcc acaatttagt
aacatttata ttaagatata 3840tatatagtgg tcactgtgat ataataaatc ataaaggaaa
ccaaatcaca aaggagatgg 3900tgtggcttag caaggaaaca gtgcaggaaa tgtaggttac
caactaagca gcttttgctc 3960ttagtactga gggatgaaag ttccagagca ttatttgaat
tctgatacat cctgccaaca 4020ctgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt
gtgtgaaaga gagacagaag 4080ggaatggttt gagagggtgc ttgtgtgcat gtgtgtgcat
atgtaaagag atttttgtgg 4140tttaagtaac tcagaatagc tgtagcaaat gactgaatac
atgtgaacaa acagaaggaa 4200gttcactctg gagtgtcttt gggaggcagc cattccaaat
gccctcctcc atttagcttc 4260aataaagggc cttttgctga tggagggcac tcaagggctg
ggtgagaggg ccacgtgttt 4320ggtattacat tactgctatg caccacttga aggagctcta
tcaccagcct caaacccgaa 4380agactgaggc attttccagt ctacttgcct aatgaatgta
taggaactgt ctatgagtat 4440ggatgtcact caactaagat caaatcacca tttaagggga
tggcattctt tatacctaaa 4500cacctaagag ctgaagtcag gtcttttaat caggttagaa
ttctaaatga tgccagagaa 4560ggcttgggaa attgtacttc agcgtgatag cctgtgtctt
cttaatttgc tgcaaaatat 4620gtggtagaga aagaaaagga aacagaaaaa tcactctggg
ttatatagca agagatgaag 4680gagaatattt caacacaggg tttttgtgtt gacataggaa
aagcctgatt cttggcaact 4740gttgtagttt gtctttcagg ggtgaaggtc ccactgacaa
cccctgttgt ggtgttccac 4800acgctgtttg ttggggtagc ttccatcggc agtctggccc
attgtcagtc atgcttcttc 4860tggccgggga gattatagag agattgtttg aagattgggt
tattattgaa agtctttttt 4920tttgtttgtt ttgttttggt ttgtttgttt atctacactt
gtttatgctg tgagccaaac 4980ctctatttaa aaagttgata ctcactttca atattttatt
tcatattatt atatatgtca 5040tgatagttat cttgatgtaa atatgaagat ttttttgttt
ctgtagatag taaactcttt 5100ttttaaaaaa ggaaaaggga aacattttta taaagttata
ttttaatcac catttttata 5160cattgtagtt ctctccaagc ccagtaagag aatgatgatt
catttgcatg gaggtcgatg 5220gacaaccaat catctacctt ttctaattta aatgataatc
tgatatagtt ttattgccag 5280ttaaatgagg atgctgcaaa gcatgttttt tcactagtaa
cttttgctaa ctgaatgaat 5340tctgggtcca tatctcccag atgaaaaact gttaaccaat
accatatttt atagttggtg 5400tccatttctt tccaacactg tttgttatga ttcttccttg
agtacttata tacagacctg 5460ctcattatct aaacaatctt accttctaag taaaccttga
ttgtgatttc cagtttttat 5520tttctctgac gtagtagaaa ggaatgttta cattaaaaat
acttttgttt ctcataaatg 5580gatattgtac tccccccttt caaagcatta ttttacaata
attcatggca ttttaaaaaa 5640taaggcaaag ataatacgac aaaaaatata catggtttca
aggcaaattc tccaataagt 5700tggaaaatgt aaaaaggatc aagtggatgc agcctctacc
taaataatta aaatatattt 5760cagtatattt ctgaattaac accaggtctt cattatttag
aacttactaa attgttttca 5820ttttcttagt tttacctgtg tatctccatg tttgcaaaaa
ttactataag tcaaattttg 5880ccagtgaatt taactatttt tctttccttg caattaaggg
gaaaaaagca tttatcttat 5940cttctcatac cccttgcatc taagtactta gcaaagtcaa
tattttccca ttttccaaat 6000gcgtccatct ctaacataaa tattaattga acatagagct
atgtttggag tgagtggact 6060ggcaggacag ttggaagtcc atcacagtct attgacagtt
tcatcaaagc tgtatagtcc 6120aactagtggg gcagcttggc tactatggtg gaagtctcag
caaactgcct ggttttgttt 6180gtttgttttg ttttaaggta caggaaataa gaggaataat
agtggccaaa gcaattagaa 6240catcttcatt ccagaactgt gttcagcaat ccaggcagat
tgatacattt ttctttaaaa 6300ataaattgct attacagcta gacgtcaatt gggataaata
aagggatgaa gatccactaa 6360gtttgtgact ttcatacaca cccagtacat ctcaaaggat
gctaagggac attttctgcc 6420agtagagttc tccccctttt tggtgacagc aatattatta
tgttcacatc taactccaga 6480gcttacttcc tgtggtgcca atgtatttgt tgcaatttac
tacattttta tatgagccta 6540tttataggtg ccattaaact caggtctttc aaatgaaaga
gtttctagcc cacttaggga 6600aaaagataat tgtttagaaa accataaaat caatggtagg
aaaagttgga actggttacc 6660tggatgccat ggttctctgt taaataaagt aagagaccag
gtgtattctg agtgtcatca 6720gtgttatttt cagcatgcta ataaatgtct ttccggttat
atatctatct aaattaacct 6780ttaaaatatt ggtttccttg ataaaagcac cacttttgct
tttgttagct gtaatatttt 6840ttgtcattta gataagacct ggtttggctc tcaataaaag
atgaagacag tagctctgta 6900cagggatata tctatattag tcttcatctg atgaatgaag
aaattttctc atattatgtt 6960caagaaagta tttacttcct aaaaatagaa ttcccgattc
tgtctatttt ggttgaatac 7020cagaacaaat ctttccgttg caatcccagt aaaacgaaag
aaaaggaata tcttacagac 7080tgttcatatt agatgtatgt agactgttaa tttgcaattt
ccccatattt cctgcctatc 7140ttacccagat aactttcttt gaaggtaaaa gctgtgcaaa
aggcatgaga ctcaggccta 7200ctctttgttt aaatgatgga aaaatataaa ttattttcta
agtaataaaa gtataaaaat 7260tatcattata aataaagtct aaagtttgaa attattaatt
taaaaaaaaa aaaaaaaaa 731921141PRTHomo sapiens 2Met Ala Val Pro Gly Asp
Ala Ala Arg Val Arg Asp Lys Pro Val His1 5
10 15Ser Gly Val Ser Gln Ala Pro Thr Ala Gly Arg Asp
Cys His His Arg 20 25 30Ala
Asp Pro Ala Ser Pro Arg Asp Ser Gly Cys Arg Gly Cys Trp Gly 35
40 45Asp Leu Val Leu Gln Pro Leu Arg Ser
Ser Arg Lys Leu Ser Ser Ala 50 55
60Leu Cys Ala Gly Ser Leu Ser Phe Leu Leu Ala Leu Leu Val Arg Leu65
70 75 80Val Arg Gly Glu Val
Gly Cys Asp Leu Glu Gln Cys Lys Glu Ala Ala 85
90 95Ala Ala Glu Glu Glu Glu Ala Ala Pro Gly Ala
Glu Gly Gly Val Phe 100 105
110Pro Gly Pro Arg Gly Gly Ala Pro Gly Gly Gly Ala Arg Leu Ser Pro
115 120 125Trp Leu Gln Pro Ser Ala Leu
Leu Phe Ser Leu Leu Cys Ala Phe Phe 130 135
140Trp Met Gly Leu Tyr Leu Leu Arg Ala Gly Val Arg Leu Pro Leu
Ala145 150 155 160Val Ala
Leu Leu Ala Ala Cys Cys Gly Gly Glu Ala Leu Val Gln Ile
165 170 175Gly Leu Gly Val Gly Glu Asp
His Leu Leu Ser Leu Pro Ala Ala Gly 180 185
190Val Val Leu Ser Cys Leu Ala Ala Ala Thr Trp Leu Val Leu
Arg Leu 195 200 205Arg Leu Gly Val
Leu Met Ile Ala Leu Thr Ser Ala Val Arg Thr Val 210
215 220Ser Leu Ile Ser Leu Glu Arg Phe Lys Val Ala Trp
Arg Pro Tyr Leu225 230 235
240Ala Tyr Leu Ala Gly Val Leu Gly Ile Leu Leu Ala Arg Tyr Val Glu
245 250 255Gln Ile Leu Pro Gln
Ser Ala Glu Ala Ala Pro Arg Glu His Leu Gly 260
265 270Ser Gln Leu Ile Ala Gly Thr Lys Glu Asp Ile Pro
Val Phe Lys Arg 275 280 285Arg Arg
Arg Ser Ser Ser Val Val Ser Ala Glu Met Ser Gly Cys Ser 290
295 300Ser Lys Ser His Arg Arg Thr Ser Leu Pro Cys
Ile Pro Arg Glu Gln305 310 315
320Leu Met Gly His Ser Glu Trp Asp His Lys Arg Gly Pro Arg Gly Ser
325 330 335Gln Ser Ser Gly
Thr Ser Ile Thr Val Asp Ile Ala Val Met Gly Glu 340
345 350Ala His Gly Leu Ile Thr Asp Leu Leu Ala Asp
Pro Ser Leu Pro Pro 355 360 365Asn
Val Cys Thr Ser Leu Arg Ala Val Ser Asn Leu Leu Ser Thr Gln 370
375 380Leu Thr Phe Gln Ala Ile His Lys Pro Arg
Val Asn Pro Val Thr Ser385 390 395
400Leu Ser Glu Asn Tyr Thr Cys Ser Asp Ser Glu Glu Ser Ser Glu
Lys 405 410 415Asp Lys Leu
Ala Ile Pro Lys Arg Leu Arg Arg Ser Leu Pro Pro Gly 420
425 430Leu Leu Arg Arg Val Ser Ser Thr Trp Thr
Thr Thr Thr Ser Ala Thr 435 440
445Gly Leu Pro Thr Leu Glu Pro Ala Pro Val Arg Arg Asp Arg Ser Thr 450
455 460Ser Ile Lys Leu Gln Glu Ala Pro
Ser Ser Ser Pro Asp Ser Trp Asn465 470
475 480Asn Pro Val Met Met Thr Leu Thr Lys Ser Arg Ser
Phe Thr Ser Ser 485 490
495Tyr Ala Ile Ser Ala Ala Asn His Val Lys Ala Lys Lys Gln Ser Arg
500 505 510Pro Gly Ala Leu Ala Lys
Ile Ser Pro Leu Ser Ser Pro Cys Ser Ser 515 520
525Pro Leu Gln Gly Thr Pro Ala Ser Ser Leu Val Ser Lys Ile
Ser Ala 530 535 540Val Gln Phe Pro Glu
Ser Ala Asp Thr Thr Ala Lys Gln Ser Leu Gly545 550
555 560Ser His Arg Ala Leu Thr Tyr Thr Gln Ser
Ala Pro Asp Leu Ser Pro 565 570
575Gln Ile Leu Thr Pro Pro Val Ile Cys Ser Ser Cys Gly Arg Pro Tyr
580 585 590Ser Gln Gly Asn Pro
Ala Asp Glu Pro Leu Glu Arg Ser Gly Val Ala 595
600 605Thr Arg Thr Pro Ser Arg Thr Asp Asp Thr Ala Gln
Val Thr Ser Asp 610 615 620Tyr Glu Thr
Asn Asn Asn Ser Asp Ser Ser Asp Ile Val Gln Asn Glu625
630 635 640Asp Glu Thr Glu Cys Leu Arg
Glu Pro Leu Arg Lys Ala Ser Ala Cys 645
650 655Ser Thr Tyr Ala Pro Glu Thr Met Met Phe Leu Asp
Lys Pro Ile Leu 660 665 670Ala
Pro Glu Pro Leu Val Met Asp Asn Leu Asp Ser Ile Met Glu Gln 675
680 685Leu Asn Thr Trp Asn Phe Pro Ile Phe
Asp Leu Val Glu Asn Ile Gly 690 695
700Arg Lys Cys Gly Arg Ile Leu Ser Gln Val Ser Tyr Arg Leu Phe Glu705
710 715 720Asp Met Gly Leu
Phe Glu Ala Phe Lys Ile Pro Ile Arg Glu Phe Met 725
730 735Asn Tyr Phe His Ala Leu Glu Ile Gly Tyr
Arg Asp Ile Pro Tyr His 740 745
750Asn Arg Ile His Ala Thr Asp Val Leu His Ala Val Trp Tyr Leu Thr
755 760 765Thr Gln Pro Ile Pro Gly Leu
Ser Thr Val Ile Asn Asp His Gly Ser 770 775
780Thr Ser Asp Ser Asp Ser Asp Ser Gly Phe Thr His Gly His Met
Gly785 790 795 800Tyr Val
Phe Ser Lys Thr Tyr Asn Val Thr Asp Asp Lys Tyr Gly Cys
805 810 815Leu Ser Gly Asn Ile Pro Ala
Leu Glu Leu Met Ala Leu Tyr Val Ala 820 825
830Ala Ala Met His Asp Tyr Asp His Pro Gly Arg Thr Asn Ala
Phe Leu 835 840 845Val Ala Thr Ser
Ala Pro Gln Ala Val Leu Tyr Asn Asp Arg Ser Val 850
855 860Leu Glu Asn His His Ala Ala Ala Ala Trp Asn Leu
Phe Met Ser Arg865 870 875
880Pro Glu Tyr Asn Phe Leu Ile Asn Leu Asp His Val Glu Phe Lys His
885 890 895Phe Arg Phe Leu Val
Ile Glu Ala Ile Leu Ala Thr Asp Leu Lys Lys 900
905 910His Phe Asp Phe Val Ala Lys Phe Asn Gly Lys Val
Asn Asp Asp Val 915 920 925Gly Ile
Asp Trp Thr Asn Glu Asn Asp Arg Leu Leu Val Cys Gln Met 930
935 940Cys Ile Lys Leu Ala Asp Ile Asn Gly Pro Ala
Lys Cys Lys Glu Leu945 950 955
960His Leu Gln Trp Thr Asp Gly Ile Val Asn Glu Phe Tyr Glu Gln Gly
965 970 975Asp Glu Glu Ala
Ser Leu Gly Leu Pro Ile Ser Pro Phe Met Asp Arg 980
985 990Ser Ala Pro Gln Leu Ala Asn Leu Gln Glu Ser
Phe Ile Ser His Ile 995 1000
1005Val Gly Pro Leu Cys Asn Ser Tyr Asp Ser Ala Gly Leu Met Pro
1010 1015 1020Gly Lys Trp Val Glu Asp
Ser Asp Glu Ser Gly Asp Thr Asp Asp 1025 1030
1035Pro Glu Glu Glu Glu Glu Glu Ala Pro Ala Pro Asn Glu Glu
Glu 1040 1045 1050Thr Cys Glu Asn Asn
Glu Ser Pro Lys Lys Lys Thr Phe Lys Arg 1055 1060
1065Arg Lys Ile Tyr Cys Gln Ile Thr Gln His Leu Leu Gln
Asn His 1070 1075 1080Lys Met Trp Lys
Lys Val Ile Glu Glu Glu Gln Arg Leu Ala Gly 1085
1090 1095Ile Glu Asn Gln Ser Leu Asp Gln Thr Pro Gln
Ser His Ser Ser 1100 1105 1110Glu Gln
Ile Gln Ala Ile Lys Glu Glu Glu Glu Glu Lys Gly Lys 1115
1120 1125Pro Arg Gly Glu Glu Ile Pro Thr Gln Lys
Pro Asp Gln 1130 1135 11403819PRTHomo
sapiens 3Met Val Thr Ile Phe Ser Lys Ser Trp Ser Phe Tyr Trp Glu Lys Ser1
5 10 15Ser Gly Thr Ser
Ile Thr Val Asp Ile Ala Val Met Gly Glu Ala His 20
25 30Gly Leu Ile Thr Asp Leu Leu Ala Asp Pro Ser
Leu Pro Pro Asn Val 35 40 45Cys
Thr Ser Leu Arg Ala Val Ser Asn Leu Leu Ser Thr Gln Leu Thr 50
55 60Phe Gln Ala Ile His Lys Pro Arg Val Asn
Pro Val Thr Ser Leu Ser65 70 75
80Glu Asn Tyr Thr Cys Ser Asp Ser Glu Glu Ser Ser Glu Lys Asp
Lys 85 90 95Leu Ala Ile
Pro Lys Arg Leu Arg Arg Ser Leu Pro Pro Gly Leu Leu 100
105 110Arg Arg Val Ser Ser Thr Trp Thr Thr Thr
Thr Ser Ala Thr Gly Leu 115 120
125Pro Thr Leu Glu Pro Ala Pro Val Arg Arg Asp Arg Ser Thr Ser Ile 130
135 140Lys Leu Gln Glu Ala Pro Ser Ser
Ser Pro Asp Ser Trp Asn Asn Pro145 150
155 160Val Met Met Thr Leu Thr Lys Ser Arg Ser Phe Thr
Ser Ser Tyr Ala 165 170
175Ile Ser Ala Ala Asn His Val Lys Ala Lys Lys Gln Ser Arg Pro Gly
180 185 190Ala Leu Ala Lys Ile Ser
Pro Leu Ser Ser Pro Cys Ser Ser Pro Leu 195 200
205Gln Gly Thr Pro Ala Ser Ser Leu Val Ser Lys Ile Ser Ala
Val Gln 210 215 220Phe Pro Glu Ser Ala
Asp Thr Thr Ala Lys Gln Ser Leu Gly Ser His225 230
235 240Arg Ala Leu Thr Tyr Thr Gln Ser Ala Pro
Asp Leu Ser Pro Gln Ile 245 250
255Leu Thr Pro Pro Val Ile Cys Ser Ser Cys Gly Arg Pro Tyr Ser Gln
260 265 270Gly Asn Pro Ala Asp
Glu Pro Leu Glu Arg Ser Gly Val Ala Thr Arg 275
280 285Thr Pro Ser Arg Thr Asp Asp Thr Ala Gln Val Thr
Ser Asp Tyr Glu 290 295 300Thr Asn Asn
Asn Ser Asp Ser Ser Asp Ile Val Gln Asn Glu Asp Glu305
310 315 320Thr Glu Cys Leu Arg Glu Pro
Leu Arg Lys Ala Ser Ala Cys Ser Thr 325
330 335Tyr Ala Pro Glu Thr Met Met Phe Leu Asp Lys Pro
Ile Leu Ala Pro 340 345 350Glu
Pro Leu Val Met Asp Asn Leu Asp Ser Ile Met Glu Gln Leu Asn 355
360 365Thr Trp Asn Phe Pro Ile Phe Asp Leu
Val Glu Asn Ile Gly Arg Lys 370 375
380Cys Gly Arg Ile Leu Ser Gln Val Ser Tyr Arg Leu Phe Glu Asp Met385
390 395 400Gly Leu Phe Glu
Ala Phe Lys Ile Pro Ile Arg Glu Phe Met Asn Tyr 405
410 415Phe His Ala Leu Glu Ile Gly Tyr Arg Asp
Ile Pro Tyr His Asn Arg 420 425
430Ile His Ala Thr Asp Val Leu His Ala Val Trp Tyr Leu Thr Thr Gln
435 440 445Pro Ile Pro Gly Leu Ser Thr
Val Ile Asn Asp His Gly Ser Thr Ser 450 455
460Asp Ser Asp Ser Asp Ser Gly Phe Thr His Gly His Met Gly Tyr
Val465 470 475 480Phe Ser
Lys Thr Tyr Asn Val Thr Asp Asp Lys Tyr Gly Cys Leu Ser
485 490 495Gly Asn Ile Pro Ala Leu Glu
Leu Met Ala Leu Tyr Val Ala Ala Ala 500 505
510Met His Asp Tyr Asp His Pro Gly Arg Thr Asn Ala Phe Leu
Val Ala 515 520 525Thr Ser Ala Pro
Gln Ala Val Leu Tyr Asn Asp Arg Ser Val Leu Glu 530
535 540Asn His His Ala Ala Ala Ala Trp Asn Leu Phe Met
Ser Arg Pro Glu545 550 555
560Tyr Asn Phe Leu Ile Asn Leu Asp His Val Glu Phe Lys His Phe Arg
565 570 575Phe Leu Val Ile Glu
Ala Ile Leu Ala Thr Asp Leu Lys Lys His Phe 580
585 590Asp Phe Val Ala Lys Phe Asn Gly Lys Val Asn Asp
Asp Val Gly Ile 595 600 605Asp Trp
Thr Asn Glu Asn Asp Arg Leu Leu Val Cys Gln Met Cys Ile 610
615 620Lys Leu Ala Asp Ile Asn Gly Pro Ala Lys Cys
Lys Glu Leu His Leu625 630 635
640Gln Trp Thr Asp Gly Ile Val Asn Glu Phe Tyr Glu Gln Gly Asp Glu
645 650 655Glu Ala Ser Leu
Gly Leu Pro Ile Ser Pro Phe Met Asp Arg Ser Ala 660
665 670Pro Gln Leu Ala Asn Leu Gln Glu Ser Phe Ile
Ser His Ile Val Gly 675 680 685Pro
Leu Cys Asn Ser Tyr Asp Ser Ala Gly Leu Met Pro Gly Lys Trp 690
695 700Val Glu Asp Ser Asp Glu Ser Gly Asp Thr
Asp Asp Pro Glu Glu Glu705 710 715
720Glu Glu Glu Ala Pro Ala Pro Asn Glu Glu Glu Thr Cys Glu Asn
Asn 725 730 735Glu Ser Pro
Lys Lys Lys Thr Phe Lys Arg Arg Lys Ile Tyr Cys Gln 740
745 750Ile Thr Gln His Leu Leu Gln Asn His Lys
Met Trp Lys Lys Val Ile 755 760
765Glu Glu Glu Gln Arg Leu Ala Gly Ile Glu Asn Gln Ser Leu Asp Gln 770
775 780Thr Pro Gln Ser His Ser Ser Glu
Gln Ile Gln Ala Ile Lys Glu Glu785 790
795 800Glu Glu Glu Lys Gly Lys Pro Arg Gly Glu Glu Ile
Pro Thr Gln Lys 805 810
815Pro Asp Gln46091DNAHomo sapiens 4gctcgcgcgc ccaacggacc aggctggggc
cgtgaggtaa ctgttgcagc cagcggaggt 60gggaggcgac actgagtctc cagtcccgag
aggtgcccga gggaaaagga ggcggcagct 120aaactggtcc tggagagaag ccccttccgc
ccctctcctc agccagcatg tcccggactc 180cgccgctcct cagtccgcgc ggtggggacc
ccgggccgtg gcggccggcg cagccctgac 240gggttgcgaa ccagggggcg ccccgaacgc
gggggttggg gtctgggagc gcgagcggcc 300gctacggtac gagcggggtg tgctgagtcc
cgtggccacc cccggcccca gccatgagga 360gggacgagcg agacgccaaa gccatgcggt
ccctgcagcc gccggatggg gccggctcgc 420cccccgagag tctgaggaac ggctacgtga
agagctgcgt gagccccttg cggcaggacc 480ctccgcgcgg cttcttcttc cacctctgcc
gcttctgcaa cgtggagctg cggccgccgc 540cggcctctcc ccagcagccg cggcgctgct
cccccttctg ccgggcgcgc ctctcgctgg 600gcgccctggc tgcctttgtc ctcgccctgc
tgctgggcgc ggaacccgag agctgggctg 660ccggggccgc ctggctgcgg acgctgctga
gcgtgtgttc gcacagcttg agccccctct 720tcagcatcgc ctgtgccttc ttcttcctca
cctgcttcct cacccggacc aagcggggac 780ccggcccggg ccggagctgc ggctcctggt
ggctgctggc gctgcccgcc tgctgttacc 840tgggggactt cttggtgtgg cagtggtggt
cttggccttg gggggatggc gacgcagggt 900ccgcggcccc gcacacgccc ccggaggcgg
cagcgggcag gttgctgctg gtgctgagct 960gcgtagggct gctgctgacg ctcgcgcacc
cgctgcggct ccggcactgc gttctggtgc 1020tgctcctggc cagcttcgtc tggtgggtct
ccttcaccag cctcgggtcg ctgccctccg 1080ccctcaggcc gctgctctcc ggcctggtgg
ggggcgctgg ctgcctgctg gccctggggt 1140tggatcactt ctttcaaatc agggaagcgc
ctcttcatcc tcgactgtcc agtgccgccg 1200aagaaaaagt gcctgtgatc cgaccccgga
ggaggtccag ctgcgtgtcg ttaggagaaa 1260ctgcagccag ttactatggc agttgcaaaa
tattcaggag accgtcgttg ccttgtattt 1320ccagagaaca gatgattctt tgggattggg
acttaaaaca atggtataag cctcattatc 1380aaaattctgg aggtggaaat ggagttgatc
tttcagtgct aaatgaggct cgcaatatgg 1440tgtcagatct tctgactgat ccaagccttc
caccacaagt catttcctct ctacggagta 1500ttagtagctt aatgggtgct ttctcaggtt
cctgtaggcc aaagattaat cctctcacac 1560catttcctgg attttacccc tgttctgaaa
tagaggaccc agctgagaaa ggggatagaa 1620aacttaacaa gggactaaat aggaatagtt
tgccaactcc acagctgagg agaagctcag 1680gaacttcagg attgctacct gttgaacagt
cttcaaggtg ggatcgtaat aatggcaaaa 1740gacctcacca agaatttggc atttcaagtc
aaggatgcta tctaaatggg ccttttaatt 1800caaatctact gactatcccg aagcaaaggt
catcttctgt atcactgact caccatgtag 1860gtctcagaag agctggtgtt ttgtccagtc
tgagtcctgt gaattcttcc aaccatggac 1920cagtgtctac tggctctcta actaatcgat
cacccataga atttcctgat actgctgatt 1980ttcttaataa gccaagcgtt atcttgcaga
gatctctggg caatgcacct aatactccag 2040atttttatca gcaacttaga aattctgata
gcaatctgtg taacagctgt ggacatcaaa 2100tgctgaaata tgtttcaaca tctgaatcag
atggtacaga ttgctgcagt ggaaaatcag 2160gtgaagaaga aaacattttc tcgaaagaat
cattcaaact tatggaaact caacaagaag 2220aggaaacaga gaagaaagac agcagaaaat
tatttcagga aggtgataag tggctaacag 2280aagaggcaca gagtgaacag caaacaaata
ttgaacagga agtatcactg gacctgattt 2340tagtagaaga gtatgactca ttaatagaaa
agatgagcaa ctggaatttt ccaatttttg 2400aacttgtaga aaagatggga gagaaatcag
gaaggattct cagtcaggtt atgtatacct 2460tatttcaaga cactggttta ttggaaatat
ttaaaattcc cactcaacaa tttatgaact 2520attttcgtgc attagaaaat ggctatcgag
acattcctta tcacaatcgt atacatgcca 2580cagatgtgct acatgcagtt tggtatctga
caacacggcc agttcctggc ttacagcaga 2640tccacaatgg ttgtggaaca ggaaatgaaa
cagattctga tggtagaatt aaccatgggc 2700gaattgctta tatttcttcg aagagctgct
ctaatcctga tgagagttat ggctgcctgt 2760cttcaaacat tcctgcatta gaattgatgg
ctctatacgt ggcagctgcc atgcatgatt 2820atgatcaccc agggaggaca aatgcatttc
tagtggctac aaatgcccct caggcagttt 2880tatacaatga cagatctgtt ctggaaaatc
atcatgctgc gtcagcttgg aatctatatc 2940tttctcgccc agaatacaac ttccttcttc
atcttgatca tgtggaattc aagcgctttc 3000gttttttagt cattgaagca atccttgcta
cggatcttaa aaagcatttt gattttctcg 3060cagaattcaa tgccaaggca aatgatgtaa
atagtaatgg catagaatgg agtaatgaaa 3120atgatcgcct cttggtatgc caggtgtgca
tcaaactggc agatataaat ggcccagcaa 3180aagttcgaga cttgcatttg aaatggacag
aaggcattgt caatgaattt tatgagcagg 3240gagatgaaga agcaaatctt ggtctgccca
tcagtccatt catggatcgt tcttctcctc 3300aactagcaaa actccaagaa tcttttatca
cccacatagt gggtcccctg tgtaactcct 3360atgatgctgc tggtttgcta ccaggtcagt
ggttagaagc agaagaggat aatgatactg 3420aaagtggtga tgatgaagac ggtgaagaat
tagatacaga agatgaagaa atggaaaaca 3480atctaaatcc aaaaccacca agaaggaaaa
gcagacggcg aatattttgt cagctaatgc 3540accacctcac tgaaaaccac aagatatgga
aggaaatcgt agaggaagaa gaaaaatgta 3600aagctgatgg gaataaactg caggtggaga
attcctcctt acctcaagca gatgagattc 3660aggtaattga agaggcagat gaagaggaat
agcgacagtt tgagtaaaag aaaagtcata 3720ttgaagaagc ccagagggtt gtgcccaggg
gcagaaatca ttgcctagtg ttcaccggct 3780gactctcaac tgaccattcc catgtggaca
ggccttaata ctgtgagagg atccttgctc 3840tgctggcagt ttcccactcc tatgcacttt
cacaggaact agaaaactat tcttaaacca 3900aaaataccat ccgtgttgac ccatgttgca
gagcccttac ttaaatcctt cactggtgta 3960tgaatacttt gtcataatgc tgctttgctg
ggtagtgagc tcttattttt cactgggggt 4020cagctataac taaaaactca agtgacatat
ttcagttacc aaagtggcca ggaacttttt 4080gcttttatga aaatagattc atattgtatt
tcccagtgtg tcttttatgt ctttgaatgt 4140tttggagaaa agtctatgcc tgtctaaaaa
tgaatccagt gttgcctttc tgagggattt 4200ctgctcaatg caatacactg ttcagtgcta
ttctcccagc taggtttatc catgaaggac 4260tgagtgacct ttgttgtatt taacaaaatc
caggtgcatc aatttctgat gctttttact 4320attgtgtatt atctactatg tgtgttttat
ttctgctgag agtattcagg tttgccatgg 4380acatcagaag tttgaattcc agtcttatct
tatgttccat ggctgaattt taaagctgtt 4440taggtttaac aatgaaggga tttattcttt
agtcaaaatt gttgttttta ctctagctca 4500ggattcgtat ttttaaagat ttagttaata
tgaacacagc acagatttgt tagaagaaaa 4560aaaatttgct gtaataccaa aactaacctc
atcaaagata cagaaaaaaa gaaatatagt 4620gagccctaaa ggacacatac attgaataaa
taattggaac atgtggttat ctttagatcc 4680acatcttagc tgtcatttgt tcactctaaa
actgatgttc atctttctgt taatttccct 4740ctgcctaaag actacatgac agaaatgacc
tatcactact tattatttct gaagcctaac 4800tgcaagactg atttctgaga acaagtaaag
aactggaata cttatttttc atataaaaat 4860ctaaatgtgt taataaatca tttcatacaa
aagtacatta ttaaataacc acattattaa 4920aataattgca agaaaatgga ccatatttac
aatgttttgt aaacttgcta gtgtgtggat 4980atgtacccta cttgtgaaat acatttgaag
atataaagag cagccaaaat gatggcaaaa 5040tggtaggcta atattttcta ttattattgg
agaacatatc atattttgga atcatgcaat 5100tttgcacaca gtgaaaccat taattttcca
aggtaattcc tttagaatat ggtattggca 5160tgcagtttct tacttatcta gaatatttgg
cttatctgaa agatatcaat ttaagatctc 5220tggaagtgtt agaatttttg atccttcaca
gtgtcaatat ttaatgaatc actaagcttt 5280atttattaga cgtgttgagt gagtgctgag
ttccttgctg ccacttttgt taccattgtc 5340acacactatg tgtaaaccag tcccaccact
tattactaat aaaattttga ctgataattt 5400atatttgcac ttacaatata tatatcctgt
ccttatattt ctctagagta cattttccat 5460catgtttaag tgtatttctg ctattatttc
ctctcctgca gaatacatac aagtgtatgt 5520gtataaagtc atacatgtac aagcatgcat
attgagattg aatcacattt ccatactgtc 5580tgttatttta ttgggtttta tattgggttt
ctttagttta tgttgttttc tcaaaagcag 5640cattttaaat tacgaatact ggacttattg
gatttaatta taaatccaat tactactgga 5700aactcatttt tacataatat agtccttaaa
ttatttaacc cttgctaagt aattgacata 5760tgtaacaata actagcctaa agaaacccaa
aaaagtatct ctcccgagct gaaacttaaa 5820aattcgtaag tgtaagaaag aatgtgagaa
tatattaaat gcacactgta ccattagatg 5880aaatcttact tgagaaattg ccataagcca
tattacagat cttactttgt tactgaatca 5940gattaatttc ttgttataat aattttcatc
ataaattttc tatttttaaa gccgctggta 6000ctagaaatat tcttttaatg ctatatctat
gtacctactg acacattttt ctccataaaa 6060gtacttttaa aaattacttc atgatttgaa a
609151112PRTHomo sapiens 5Met Arg Arg
Asp Glu Arg Asp Ala Lys Ala Met Arg Ser Leu Gln Pro1 5
10 15Pro Asp Gly Ala Gly Ser Pro Pro Glu
Ser Leu Arg Asn Gly Tyr Val 20 25
30Lys Ser Cys Val Ser Pro Leu Arg Gln Asp Pro Pro Arg Gly Phe Phe
35 40 45Phe His Leu Cys Arg Phe Cys
Asn Val Glu Leu Arg Pro Pro Pro Ala 50 55
60Ser Pro Gln Gln Pro Arg Arg Cys Ser Pro Phe Cys Arg Ala Arg Leu65
70 75 80Ser Leu Gly Ala
Leu Ala Ala Phe Val Leu Ala Leu Leu Leu Gly Ala 85
90 95Glu Pro Glu Ser Trp Ala Ala Gly Ala Ala
Trp Leu Arg Thr Leu Leu 100 105
110Ser Val Cys Ser His Ser Leu Ser Pro Leu Phe Ser Ile Ala Cys Ala
115 120 125Phe Phe Phe Leu Thr Cys Phe
Leu Thr Arg Thr Lys Arg Gly Pro Gly 130 135
140Pro Gly Arg Ser Cys Gly Ser Trp Trp Leu Leu Ala Leu Pro Ala
Cys145 150 155 160Cys Tyr
Leu Gly Asp Phe Leu Val Trp Gln Trp Trp Ser Trp Pro Trp
165 170 175Gly Asp Gly Asp Ala Gly Ser
Ala Ala Pro His Thr Pro Pro Glu Ala 180 185
190Ala Ala Gly Arg Leu Leu Leu Val Leu Ser Cys Val Gly Leu
Leu Leu 195 200 205Thr Leu Ala His
Pro Leu Arg Leu Arg His Cys Val Leu Val Leu Leu 210
215 220Leu Ala Ser Phe Val Trp Trp Val Ser Phe Thr Ser
Leu Gly Ser Leu225 230 235
240Pro Ser Ala Leu Arg Pro Leu Leu Ser Gly Leu Val Gly Gly Ala Gly
245 250 255Cys Leu Leu Ala Leu
Gly Leu Asp His Phe Phe Gln Ile Arg Glu Ala 260
265 270Pro Leu His Pro Arg Leu Ser Ser Ala Ala Glu Glu
Lys Val Pro Val 275 280 285Ile Arg
Pro Arg Arg Arg Ser Ser Cys Val Ser Leu Gly Glu Thr Ala 290
295 300Ala Ser Tyr Tyr Gly Ser Cys Lys Ile Phe Arg
Arg Pro Ser Leu Pro305 310 315
320Cys Ile Ser Arg Glu Gln Met Ile Leu Trp Asp Trp Asp Leu Lys Gln
325 330 335Trp Tyr Lys Pro
His Tyr Gln Asn Ser Gly Gly Gly Asn Gly Val Asp 340
345 350Leu Ser Val Leu Asn Glu Ala Arg Asn Met Val
Ser Asp Leu Leu Thr 355 360 365Asp
Pro Ser Leu Pro Pro Gln Val Ile Ser Ser Leu Arg Ser Ile Ser 370
375 380Ser Leu Met Gly Ala Phe Ser Gly Ser Cys
Arg Pro Lys Ile Asn Pro385 390 395
400Leu Thr Pro Phe Pro Gly Phe Tyr Pro Cys Ser Glu Ile Glu Asp
Pro 405 410 415Ala Glu Lys
Gly Asp Arg Lys Leu Asn Lys Gly Leu Asn Arg Asn Ser 420
425 430Leu Pro Thr Pro Gln Leu Arg Arg Ser Ser
Gly Thr Ser Gly Leu Leu 435 440
445Pro Val Glu Gln Ser Ser Arg Trp Asp Arg Asn Asn Gly Lys Arg Pro 450
455 460His Gln Glu Phe Gly Ile Ser Ser
Gln Gly Cys Tyr Leu Asn Gly Pro465 470
475 480Phe Asn Ser Asn Leu Leu Thr Ile Pro Lys Gln Arg
Ser Ser Ser Val 485 490
495Ser Leu Thr His His Val Gly Leu Arg Arg Ala Gly Val Leu Ser Ser
500 505 510Leu Ser Pro Val Asn Ser
Ser Asn His Gly Pro Val Ser Thr Gly Ser 515 520
525Leu Thr Asn Arg Ser Pro Ile Glu Phe Pro Asp Thr Ala Asp
Phe Leu 530 535 540Asn Lys Pro Ser Val
Ile Leu Gln Arg Ser Leu Gly Asn Ala Pro Asn545 550
555 560Thr Pro Asp Phe Tyr Gln Gln Leu Arg Asn
Ser Asp Ser Asn Leu Cys 565 570
575Asn Ser Cys Gly His Gln Met Leu Lys Tyr Val Ser Thr Ser Glu Ser
580 585 590Asp Gly Thr Asp Cys
Cys Ser Gly Lys Ser Gly Glu Glu Glu Asn Ile 595
600 605Phe Ser Lys Glu Ser Phe Lys Leu Met Glu Thr Gln
Gln Glu Glu Glu 610 615 620Thr Glu Lys
Lys Asp Ser Arg Lys Leu Phe Gln Glu Gly Asp Lys Trp625
630 635 640Leu Thr Glu Glu Ala Gln Ser
Glu Gln Gln Thr Asn Ile Glu Gln Glu 645
650 655Val Ser Leu Asp Leu Ile Leu Val Glu Glu Tyr Asp
Ser Leu Ile Glu 660 665 670Lys
Met Ser Asn Trp Asn Phe Pro Ile Phe Glu Leu Val Glu Lys Met 675
680 685Gly Glu Lys Ser Gly Arg Ile Leu Ser
Gln Val Met Tyr Thr Leu Phe 690 695
700Gln Asp Thr Gly Leu Leu Glu Ile Phe Lys Ile Pro Thr Gln Gln Phe705
710 715 720Met Asn Tyr Phe
Arg Ala Leu Glu Asn Gly Tyr Arg Asp Ile Pro Tyr 725
730 735His Asn Arg Ile His Ala Thr Asp Val Leu
His Ala Val Trp Tyr Leu 740 745
750Thr Thr Arg Pro Val Pro Gly Leu Gln Gln Ile His Asn Gly Cys Gly
755 760 765Thr Gly Asn Glu Thr Asp Ser
Asp Gly Arg Ile Asn His Gly Arg Ile 770 775
780Ala Tyr Ile Ser Ser Lys Ser Cys Ser Asn Pro Asp Glu Ser Tyr
Gly785 790 795 800Cys Leu
Ser Ser Asn Ile Pro Ala Leu Glu Leu Met Ala Leu Tyr Val
805 810 815Ala Ala Ala Met His Asp Tyr
Asp His Pro Gly Arg Thr Asn Ala Phe 820 825
830Leu Val Ala Thr Asn Ala Pro Gln Ala Val Leu Tyr Asn Asp
Arg Ser 835 840 845Val Leu Glu Asn
His His Ala Ala Ser Ala Trp Asn Leu Tyr Leu Ser 850
855 860Arg Pro Glu Tyr Asn Phe Leu Leu His Leu Asp His
Val Glu Phe Lys865 870 875
880Arg Phe Arg Phe Leu Val Ile Glu Ala Ile Leu Ala Thr Asp Leu Lys
885 890 895Lys His Phe Asp Phe
Leu Ala Glu Phe Asn Ala Lys Ala Asn Asp Val 900
905 910Asn Ser Asn Gly Ile Glu Trp Ser Asn Glu Asn Asp
Arg Leu Leu Val 915 920 925Cys Gln
Val Cys Ile Lys Leu Ala Asp Ile Asn Gly Pro Ala Lys Val 930
935 940Arg Asp Leu His Leu Lys Trp Thr Glu Gly Ile
Val Asn Glu Phe Tyr945 950 955
960Glu Gln Gly Asp Glu Glu Ala Asn Leu Gly Leu Pro Ile Ser Pro Phe
965 970 975Met Asp Arg Ser
Ser Pro Gln Leu Ala Lys Leu Gln Glu Ser Phe Ile 980
985 990Thr His Ile Val Gly Pro Leu Cys Asn Ser Tyr
Asp Ala Ala Gly Leu 995 1000
1005Leu Pro Gly Gln Trp Leu Glu Ala Glu Glu Asp Asn Asp Thr Glu
1010 1015 1020Ser Gly Asp Asp Glu Asp
Gly Glu Glu Leu Asp Thr Glu Asp Glu 1025 1030
1035Glu Met Glu Asn Asn Leu Asn Pro Lys Pro Pro Arg Arg Lys
Ser 1040 1045 1050Arg Arg Arg Ile Phe
Cys Gln Leu Met His His Leu Thr Glu Asn 1055 1060
1065His Lys Ile Trp Lys Glu Ile Val Glu Glu Glu Glu Lys
Cys Lys 1070 1075 1080Ala Asp Gly Asn
Lys Leu Gln Val Glu Asn Ser Ser Leu Pro Gln 1085
1090 1095Ala Asp Glu Ile Gln Val Ile Glu Glu Ala Asp
Glu Glu Glu 1100 1105
111062530DNAHomo sapiens 6tttgtaactt cacttcagcc tcccattgat cgctttctgc
aaccattcag actgatctcg 60ggctcctatt tcatttacat tgtgtgcaca ccaagtaacc
agtgggaaaa ctttagaggg 120tacttaaacc ccagaaaatt ctgaaaccgg gctcttgagc
cgctatcctc gggcctgctc 180ccaccctgtg gagtgcactt tcgttttcaa taaatctctg
cttttgttgc ttcattcttt 240ccttgctttg tttgtgtgtt tgtccagttc tttgttcaac
acgccaagaa cctggacact 300cttcactggt aacatatttt ggcaagccaa ccaggagaaa
agaatttctg cttggacact 360gcatagctgc tgggaaaatg aacatcagtg ttgatttgga
aacgaattat gccgagttgg 420ttctagatgt gggaagagtc actcttggag agaacagtag
gaaaaaaatg aaggattgta 480aactgagaaa aaagcagaat gaaagtgtct cacgagctat
gtgtgctctg ctcaattctg 540gagggggagt gatcaaggct gaaattgaga atgaagacta
tagttataca aaagatggaa 600taggactaga tttggaaaat tcttttagta acattctgtt
atttgttcct gagtacttag 660acttcatgca gaatggtaac tactttctga tttttgtgaa
gtcatggagc ttgaacacct 720ctggtctgcg gattaccacc ttgagctcca atttgtacaa
aagagatata acatctgcaa 780aagtcatgaa tgccactgct gcactggagt tcctcaaaga
catgaaaaag actagaggga 840gattgtattt aagaccagaa ttgctggcaa agaggccctg
tgttgatata caagaagaaa 900ataacatgaa ggccttggcc ggggtttttt ttgatagaac
agaacttgat cggaaagaaa 960aattgacctt tactgaatcc acacatgttg aaattaaaaa
cttctcgaca gaaaagttgt 1020tacaacgaat taaagagatt ctccctcaat atgtttctgc
atttgcaaat actgatggag 1080gatatttgtt cattggttta aatgaagata aagaaataat
tggctttaaa gcagagatga 1140gtgacctcga tgacttagaa agagaaatcg aaaagtccat
taggaagatg cctgtgcatc 1200acttctgtat ggagaagaag aagataaatt attcatgcaa
attccttgga gtatatgata 1260aaggaagtct ttgtggatat gtctgtgcac tcagagtgga
gcgcttctgc tgtgcagtgt 1320ttgctaaaga gcctgattcc tggcatgtga aagataaccg
tgtgatgcag ttgaccagga 1380aggaatggat ccagttcatg gtggaggctg aaccaaaatt
ttccagttca tatgaagagg 1440tgatctctca aataaatacg tcattacctg ctccccacag
ttggcctctt ttggaatggc 1500aacggcagag acatcactgt ccagggctat caggaaggat
aacgtatact ccagaaaacc 1560tttgcagaaa actgttctta caacatgaag gacttaagca
attaatatgt gaagaaatgg 1620actctgtcag aaagggctca ctgatcttct ctaggagctg
gtctgtggat ctgggcttgc 1680aagagaacca caaagtcctc tgtgatgctc ttctgatttc
ccaggacagt cctccagtcc 1740tatacacctt ccacatggta caggatgagg agtttaaagg
ctattctaca caaactgccc 1800taaccttaaa gcagaagctg gcaaaaattg gtggttacac
taaaaaagtg tgtgtcatga 1860caaagatctt ctacttgagc cctgaaggca tgacaagctg
ccagtatgat ttaaggtcgc 1920aagtaattta ccctgaatcc tactatttta caagaaggaa
atacttgctg aaagcccttt 1980ttaaagcctt aaagagactc aagtctctga gagaccagtt
ttcctttgca gaaaatctat 2040accagataat cggtatagat tgctttcaga agaatgataa
aaagatgttt aaatcttgtc 2100gaaggctcac ctgatggaaa atggactggg ctactgagat
atttttcatt atatatttga 2160taacattctc taattctgtg aaaatatttc tttgaaaact
ttgcaagtta agcaacttaa 2220tgtgatgttg gataattggg ttttgtctat tttcacttct
ccctaaataa tcttcacaga 2280tattgtttga gggatattag gaaaattaat ttgttaactc
gtctgtgcac agtattattt 2340actctgtctg tagttcctga ataaattttc ttccatgctt
gaactgggaa aattgcaaca 2400cttttattct taatgacaac agtgaaaatc tcccagcata
tacctagaaa acaattataa 2460cttacaaaag attatccttg atgaaactca gaatttccac
agtgggaatg aataagaagg 2520caaaactcat
25307578PRTHomo sapiens 7Met Asn Ile Ser Val Asp
Leu Glu Thr Asn Tyr Ala Glu Leu Val Leu1 5
10 15Asp Val Gly Arg Val Thr Leu Gly Glu Asn Ser Arg
Lys Lys Met Lys 20 25 30Asp
Cys Lys Leu Arg Lys Lys Gln Asn Glu Ser Val Ser Arg Ala Met 35
40 45Cys Ala Leu Leu Asn Ser Gly Gly Gly
Val Ile Lys Ala Glu Ile Glu 50 55
60Asn Glu Asp Tyr Ser Tyr Thr Lys Asp Gly Ile Gly Leu Asp Leu Glu65
70 75 80Asn Ser Phe Ser Asn
Ile Leu Leu Phe Val Pro Glu Tyr Leu Asp Phe 85
90 95Met Gln Asn Gly Asn Tyr Phe Leu Ile Phe Val
Lys Ser Trp Ser Leu 100 105
110Asn Thr Ser Gly Leu Arg Ile Thr Thr Leu Ser Ser Asn Leu Tyr Lys
115 120 125Arg Asp Ile Thr Ser Ala Lys
Val Met Asn Ala Thr Ala Ala Leu Glu 130 135
140Phe Leu Lys Asp Met Lys Lys Thr Arg Gly Arg Leu Tyr Leu Arg
Pro145 150 155 160Glu Leu
Leu Ala Lys Arg Pro Cys Val Asp Ile Gln Glu Glu Asn Asn
165 170 175Met Lys Ala Leu Ala Gly Val
Phe Phe Asp Arg Thr Glu Leu Asp Arg 180 185
190Lys Glu Lys Leu Thr Phe Thr Glu Ser Thr His Val Glu Ile
Lys Asn 195 200 205Phe Ser Thr Glu
Lys Leu Leu Gln Arg Ile Lys Glu Ile Leu Pro Gln 210
215 220Tyr Val Ser Ala Phe Ala Asn Thr Asp Gly Gly Tyr
Leu Phe Ile Gly225 230 235
240Leu Asn Glu Asp Lys Glu Ile Ile Gly Phe Lys Ala Glu Met Ser Asp
245 250 255Leu Asp Asp Leu Glu
Arg Glu Ile Glu Lys Ser Ile Arg Lys Met Pro 260
265 270Val His His Phe Cys Met Glu Lys Lys Lys Ile Asn
Tyr Ser Cys Lys 275 280 285Phe Leu
Gly Val Tyr Asp Lys Gly Ser Leu Cys Gly Tyr Val Cys Ala 290
295 300Leu Arg Val Glu Arg Phe Cys Cys Ala Val Phe
Ala Lys Glu Pro Asp305 310 315
320Ser Trp His Val Lys Asp Asn Arg Val Met Gln Leu Thr Arg Lys Glu
325 330 335Trp Ile Gln Phe
Met Val Glu Ala Glu Pro Lys Phe Ser Ser Ser Tyr 340
345 350Glu Glu Val Ile Ser Gln Ile Asn Thr Ser Leu
Pro Ala Pro His Ser 355 360 365Trp
Pro Leu Leu Glu Trp Gln Arg Gln Arg His His Cys Pro Gly Leu 370
375 380Ser Gly Arg Ile Thr Tyr Thr Pro Glu Asn
Leu Cys Arg Lys Leu Phe385 390 395
400Leu Gln His Glu Gly Leu Lys Gln Leu Ile Cys Glu Glu Met Asp
Ser 405 410 415Val Arg Lys
Gly Ser Leu Ile Phe Ser Arg Ser Trp Ser Val Asp Leu 420
425 430Gly Leu Gln Glu Asn His Lys Val Leu Cys
Asp Ala Leu Leu Ile Ser 435 440
445Gln Asp Ser Pro Pro Val Leu Tyr Thr Phe His Met Val Gln Asp Glu 450
455 460Glu Phe Lys Gly Tyr Ser Thr Gln
Thr Ala Leu Thr Leu Lys Gln Lys465 470
475 480Leu Ala Lys Ile Gly Gly Tyr Thr Lys Lys Val Cys
Val Met Thr Lys 485 490
495Ile Phe Tyr Leu Ser Pro Glu Gly Met Thr Ser Cys Gln Tyr Asp Leu
500 505 510Arg Ser Gln Val Ile Tyr
Pro Glu Ser Tyr Tyr Phe Thr Arg Arg Lys 515 520
525Tyr Leu Leu Lys Ala Leu Phe Lys Ala Leu Lys Arg Leu Lys
Ser Leu 530 535 540Arg Asp Gln Phe Ser
Phe Ala Glu Asn Leu Tyr Gln Ile Ile Gly Ile545 550
555 560Asp Cys Phe Gln Lys Asn Asp Lys Lys Met
Phe Lys Ser Cys Arg Arg 565 570
575Leu Thr88080DNAHomo sapiens 8ttttggcttc tgccctcaac caaaatggcg
ctagctcgga agctgccgag gtgctaggag 60ttgccgaagc aagtccggaa gctaccgagc
gagtccggaa gttgccgaaa gggagcagcg 120gggaaggagg atggcggata tcatcgcaag
actccgggag gacgggatcc aaaaacgtgt 180gatacaggaa ggccgaggag agctcccgga
ctttcaagat gggaccaagg ttcgtgtcta 240ccctaccctt ctccccctct gcggcgtggt
gcgcatgcga ggcgggagga ggccttaggc 300gagaggttgc gcatgcccag agggcagcgt
ccactgcccc taccgctcac atgcagaact 360cgacgctgat tgggctgaat ttaagtaggg
ggtgaattcg ggcctgtctg ccccgccccc 420tggctcggcc ttgtagcagc attggtgggg
gaggccgtca gtcatcacaa gcgggttggg 480gttgggggtt gatctcagtg cttgggcaga
ccccacgctg gaggaaaccc agggccggga 540gtggtcctcg ggtatctggg tttcaaggct
catgatcctt tgtagatgga agggccttct 600gaaaacactt agaccaactg ccgctgttta
gagtggaaaa ccaagaccct gggacgtgca 660aagccggaga acgggcccag aggtcaggtc
tcccagacag ggactcttta gcagccttcc 720tgctgcacta ggggcttgtt gggacagatg
agggttggga agtaaagaac ctcccacttt 780tctccttttt gccaggcccc cagatccagc
ccctctgccc gcttctcccc caacctacaa 840ctccaggctt ccctgcttct cctgtagttg
cctcctcccg gagtgctttt cccagctgcc 900acttgtttgc agagtaggga acctcccagg
ggcagcccct gtgcccagca gagcagtcag 960gcaggacatg cacattgagc aaatgagcac
atgccccctg gccagcaccg tgccgaatcg 1020ggcagctaag catcctagcc cagtgcagta
taagtgccct gagagcagag gggagctgca 1080tggctggagt gatccgctgt atgaaaagat
atcttctcta agaagagaca ggatgtgtgg 1140tgtgggttca tgcccccatg tgctgggggg
ttggtggcgt tggaagaagg ggctggcaag 1200ggggatcctg gatggaacag acatcagaag
gagagatgtg aacaatggca ccccaagatc 1260agaaacaggt ggtgttaaat aaccaatcgc
cagcactgat tgagtgctca ctattcgaac 1320attgtgctac atgcttcaca cgtttatttc
ctacaatgtg agataggtac tgttgttgat 1380tccgttttac cgatgtggaa actgacttca
gagatgcagc atggtgcggc agttaagagc 1440gtgggctcct ctaaccatat cctgtcgaga
gttcaatctc caaacctctt ttctctgcac 1500ccacccccag tgttatctct aaaaactctc
cctgcccgga ttactcccag atgcagctct 1560ccagtcatta actgtctctt aaacctgata
tatagctccc tactcaccat atccacctgg 1620aagcctggtt ggcaactcac acttaacctg
ctccacctga ggcttctccg tgtcagggga 1680accaacaacc ttcccgttgt tcagggcaaa
aaccttagca tctctgtggt cctcccagtc 1740tcacatccaa catcacatcc tcaatatcca
gccaggatct gagttctcac cacttctgcc 1800atcactgctt gggtccaggc catcctcatc
tccagcctgg gttactgcag cgacctctaa 1860ctctcctgcc tcttttgtcc ctctgtggtc
tgttctcgtc ccagcagccg agcccatgcc 1920agattcaatt ccttttttgc tcggagccac
tcagtggctt ccatcacaga gtgaaaaaca 1980gaggcctcac catagcctac aggccctgtg
aggtccaccc ctactgacct gggtgagctc 2040ccctgctgac cctgtggtgt accccacccc
ctccttcact ctgctctgcc acactggcat 2100tgctgctctt gaacacatca tgcatttgaa
acgggaagtt cccttgtctc cctcgcaggg 2160cgtgcgatgg gggagtggct cgcttcttca
gtgccccgct gctcagacct ctgggggagc 2220atacagatgg gcaggctgtg ggctccgacc
tcatggcagt gtctaggggt gaatatttac 2280agctccgtgt gttctagggt gctcttttag
tttgtctatg ggaggcttgt gttaaccagc 2340tcaattagac ccccttcctt atcacaagga
cagagggctt tctgtagtct ggggttttct 2400tgccttgatg tactggagta ctggagaatt
agatcacttg tgggcttgga gaatgattgc 2460aaattttttt ttatttttta ttttattttt
tttttctgag atggagtttc actcttgttg 2520cccaggctgg agtgcaatgg cacaatctct
gcctcccagg ttcaagcaat tctcctgcct 2580cagcctccca agtagctgag attacatgtg
cctgacacca ggcccggcta atttttaaaa 2640atgtttttag tagagatggg cttttaccat
gttggccaag ctggtttcaa acgccttttt 2700tttttttttt ttttttgaga cggagtcttg
ctctattgcc caagctggag tgcagtggca 2760tgatctcggt tcactgcaac ctccaccttc
tgggttcaag tgattctcct gcctcagcct 2820cccaagtagc tgggattaca ggcacccgcc
atcatggcca gctaattttt gtatttttag 2880tagagacggg gttttgccac attggccagg
ctggtcttga actcctgacc tcaggtgatc 2940cacccgcctt ggagatggtc ttcccctggg
gttgggccac ttggtggccc cacctctcct 3000ctgactgccc cagccaaact ccgcctcttc
ctgccagttg atgacctgcc agcgtgcagg 3060tgcctgtcag tgtgatcttc tgcttcttgc
tcccctgaca tcctctcaat gaccaggagc 3120tcgtcttctg ctgatgggct cctctgacat
ctggctgcct gtgggtctac cccctagggg 3180tgttgggttt ttataggcac aggatagggg
tgtggcaggc cagggtggtc ttgggaaatg 3240caacatttgg gcaggaaatg cctgttctca
cctaggtctg tgggggtgga accctaccca 3300gggaccacgc cctcctctac ccagcacttc
ccttctcccc ttccaaatta tttaacagga 3360ccatgctcct cccttcccag cacttccata
tcacattgtc ccactgcaag gcttttttac 3420acatgctgtt cttttggcct agaaagttcc
tatcccaggg tccacttggc ttgctttctt 3480ccttactccc caacccccca ctctgtttaa
tccagcccca accctcttgc cctgctgttt 3540cccaagcacg tggcttcacc tgccatgaca
tattgttttg tttgatgccc atctcctccc 3600tctagaagcg ccatgtgagc tccagggggg
cagggacttt tttgtgtttt gcttgctgcc 3660atgttctggt gtctagcaca gagcttgggc
acatagtagg tgcttagtaa atatctgttg 3720aggaatgact ggagtcagac tgcttggact
cttgttccca ctcagccacc cactagccgt 3780gtggcttggg cctattcctc ccctccttgt
ggctttgttt tctcaccagc gtgggaggat 3840gaagccaggt gtaaggtcag gtggtgtccc
cggggaagcc ccgtccctta tgccgtctgc 3900aggccgggga ctggacttct ccttgggggt
cagggtgagg gtttgtgcct ttgcctgacc 3960tcgcatgtgg cccacaggcc acgttccact
accggacgct gcacagtgac gacgagggca 4020ccgtgctgga cgacagccgg gctcgtggca
agcccatgga gctcatcatt ggcaagaagt 4080tcaagctgcc tgtgtgggag accatcgtgt
gcaccatgcg agaaggggag attgcccagt 4140tcctctgtga catcaaggtg tctgtcctgt
acctgtctgc ggtggctgtc cagccaagcc 4200ctattcctat tccctatccc cagggctcct
cctccctcca ccctctgcta gactgccacc 4260cgctttcttt ttttttttga gatggagtct
tgctctgtcg cccaggctgg agtgcagtgg 4320tacgatctca gctcactgca ccctccacct
cctgggttca agcgattctt ctgcctcagc 4380ctcccgagta gctgggatta caaacacccg
ccatgatgcc tggctaattt ttgtattttt 4440agtagagaca gggtttcacc atgttggcca
ggctggtctt gaacacctga cctcaggtga 4500tccacccgcc ttggcctccc aaagtgctgg
gattataggc gtgtgccacc gcgcccggcc 4560cacccactct ttccagacca ccacaccagc
ctgctgatgg cgtcctggcc tccattccgc 4620cttcccctat tagccagact gaggccaggg
gactcgttct caaatgcaaa tgacctgtac 4680atccctttgt ttcaaacctc tatgactcct
ggtcactgta aggatagagc acagggggtc 4740ctcacttcat gttgctgata cattcttgga
aactgtgact aagagaaaaa acatacatca 4800ggttttttct cagccaccgt catttctctc
agcaaaattt tgttagaaca ttgatgagaa 4860gaaaaattgg tttcgttatg tattgtttcg
cctacagtca cagtttccaa gaacctactt 4920aggacgttaa gtgaggactt aaaccgtata
agctatagct gctcacatag ctttttgggg 4980gctggcccct gccgtctcac cctcttactc
aacctccctg cttttccttt ccattcccct 5040tcttagccaa gatcttccct cttccttcaa
agcttattcc tgggtcacca cctctaggaa 5100gccctccctg actgctagtg gttggctcaa
ctcccatgtt tgggtcctcc aaccctcatg 5160ccctgcatgg ccaggatctg ctcttctgcc
ttgtcctagg ctattgcaga gcagggatct 5220ggcctgttta cttctagctt tgggatgccc
agcgcgagcc agtccagagc caagactcag 5280gaaatgcccg ctgatggcag cccggcagtc
agcccctgtc cagacaacag ggcagtggga 5340ggagtgggga ggacccgggt aggaggaatc
tggttatctg gttcccacca gcctagcagc 5400tttgccaagc aagagattag aggctaggtc
ccctatgcct gtctccctgt ggggtttttt 5460tttttttgac taagtctcac tctgttgccc
aggctggagt gcagtggcgt gatcttggct 5520cactgcaacc accatctcct gggttcagct
gattctctgc cttagctgcc tgagtagctg 5580ggattacagg cacctgccat cgtgcccggc
tcatttttgt attttagcag agacgcggtt 5640tcaccatgtt ggtcaggctg gtcttgaact
cctgacctca ggtgatccgc ccgccttggc 5700ctcccgaagt gctaggatta caggcgtgag
ccaccacatc cggcctccct gagggttttg 5760aagtggctgg cctgggccca gctctgaggt
aggccctcag tggggtgtgg gtggggcaga 5820aggaggagct gctgggaaca gaatgtgggg
ggccccagtt ctttgcatag tccagcaaag 5880ggccttatcc tctggaggga gaggaggtaa
gaattctact gggcctgtaa ggaccaggga 5940gacaggggtt gatggtaggc atgtgtctgt
ggtgggggtg aggagggggt taggtgctct 6000gtttggtggc cagagaatgt ggcagaagct
ggggcttcac caggagagag ggctgagcga 6060ctggaggagt cctgaattaa aagcctcctg
tgcttaaacg gagtagggtc ccagttgtca 6120ctctctgggc cttggtgttt gttctcagat
ggtggtgggg aagggggctg ggccttgtgg 6180acccggtgac cagccagccc acggtgacag
agcccccggc gcccttgcct tcccgcagca 6240tgtggtcctg tacccgctgg tggccaagag
tctccgcaac atcgcggtgg gcaaggaccc 6300cctggagggc cagcggcact gctgcggtgt
tgcacagatg cgtgaacaca gctccctggg 6360ccatgctgac ctggacgccc tgcagcagaa
cccccagccc ctcatcttcc acatggagat 6420gctgaaggtg aggggccacc gcgcctggtc
tcaccaggcc cccactgccc agcctcaggg 6480cggcgctggc ctgtccaccc aggggtggtg
ggatccgcag gtggactgct gggggagcgg 6540acagagacaa gaaaacctgt gcaggaccct
tggcagtacc ctgggtctcc tttcctcctc 6600cttcacatct caaatgtcac ctcctccagg
aaaccggccc tgcccacccg gtctcctcat 6660tctctgtctc gcagcagctc atttccttta
tagcctctgc cgcaccttga agtcccttgg 6720aattcatgga tttccttgtc catttaggga
acctgccatg cagcatgatc tctgcgaggg 6780cagggctttt caccgtcttg ttcactgttc
tattcttagc acttggcaca gtgctgggca 6840cacaggagat gtgacatcga tgtttgatgc
tttttgagtg acaagtagct ctgctgctgg 6900tgtgtgatgt ctgggggccc agccagccca
gatgtgggtc aggtctgctg ctgacggacg 6960cagctgtggt gtccccgagc cccgctgtga
tatgccccat gccctgcagg tggagagccc 7020tggcacgtac cagcaggacc catgggccat
gacagacgaa gagaaggcaa aggcagtgcc 7080acttatccac caggagggca accggttgta
ccgcgagggg catgtgaagg aggctgctgc 7140caagtactac gatgccattg cctgcctcaa
gaacctgcag atgaaggtac tgcctggagg 7200ctgaggggga ggatggatgg aggggggtgt
ggagccaggg ggcccaggtc tacagcttct 7260ccccgctccc tgcccccata ctcccaggaa
cagcctgggt cccctgaatg gatccagctg 7320gaccagcaga tcacgccgct gctgctcaac
tactgccagt gcaagctggt ggtcgaggag 7380tactacgagg tgctggacca ctgctcttcc
atcctcaaca agtacgacgg tgagcaccgg 7440gccctgggct gccgggggct gcgagtggtc
agagagtggc ctttctcctg tcactgctgg 7500ggtcaagacc tagcctttca caacccccat
tctgagctcc cacgggggcc tgactaaatg 7560cctctactcg gcagggctgt gggccccatt
gtgccaatga agcatgaatg gtgtattggg 7620ggtggggtgg catcctcagg tcagggaggg
ctctctctcc cctgtgggcc catggtgcca 7680ggagacatga gggcaggcag ctggccagga
tcccccctca tgcccttgca tgcccactgc 7740ccactggcct cccctgcaga caacgtcaag
gcctacttca agcggggcaa ggcccacgcg 7800gccgtgtgga atgcccagga ggcccaggct
gactttgcca aagtgctgga gctggaccca 7860gccctggcgc ctgtggtgag ccgagagctg
caggccctgg aggcacggat ccggcagaag 7920gacgaagagg acaaagcccg gttccggggg
atcttctccc attgacagga gcacttggcc 7980ctgccttacc tgccaagccc actgctgcag
ctgccagccc ccctgcccgt gctgcgtcat 8040gcttctgtgt atataaaggc ctttatttat
ctctctctga 80809330PRTHomo sapiens 9Met Ala Asp
Ile Ile Ala Arg Leu Arg Glu Asp Gly Ile Gln Lys Arg1 5
10 15Val Ile Gln Glu Gly Arg Gly Glu Leu
Pro Asp Phe Gln Asp Gly Thr 20 25
30Lys Ala Thr Phe His Tyr Arg Thr Leu His Ser Asp Asp Glu Gly Thr
35 40 45Val Leu Asp Asp Ser Arg Ala
Arg Gly Lys Pro Met Glu Leu Ile Ile 50 55
60Gly Lys Lys Phe Lys Leu Pro Val Trp Glu Thr Ile Val Cys Thr Met65
70 75 80Arg Glu Gly Glu
Ile Ala Gln Phe Leu Cys Asp Ile Lys His Val Val 85
90 95Leu Tyr Pro Leu Val Ala Lys Ser Leu Arg
Asn Ile Ala Val Gly Lys 100 105
110Asp Pro Leu Glu Gly Gln Arg His Cys Cys Gly Val Ala Gln Met Arg
115 120 125Glu His Ser Ser Leu Gly His
Ala Asp Leu Asp Ala Leu Gln Gln Asn 130 135
140Pro Gln Pro Leu Ile Phe His Met Glu Met Leu Lys Val Glu Ser
Pro145 150 155 160Gly Thr
Tyr Gln Gln Asp Pro Trp Ala Met Thr Asp Glu Glu Lys Ala
165 170 175Lys Ala Val Pro Leu Ile His
Gln Glu Gly Asn Arg Leu Tyr Arg Glu 180 185
190Gly His Val Lys Glu Ala Ala Ala Lys Tyr Tyr Asp Ala Ile
Ala Cys 195 200 205Leu Lys Asn Leu
Gln Met Lys Glu Gln Pro Gly Ser Pro Glu Trp Ile 210
215 220Gln Leu Asp Gln Gln Ile Thr Pro Leu Leu Leu Asn
Tyr Cys Gln Cys225 230 235
240Lys Leu Val Val Glu Glu Tyr Tyr Glu Val Leu Asp His Cys Ser Ser
245 250 255Ile Leu Asn Lys Tyr
Asp Asp Asn Val Lys Ala Tyr Phe Lys Arg Gly 260
265 270Lys Ala His Ala Ala Val Trp Asn Ala Gln Glu Ala
Gln Ala Asp Phe 275 280 285Ala Lys
Val Leu Glu Leu Asp Pro Ala Leu Ala Pro Val Val Ser Arg 290
295 300Glu Leu Gln Ala Leu Glu Ala Arg Ile Arg Gln
Lys Asp Glu Glu Asp305 310 315
320Lys Ala Arg Phe Arg Gly Ile Phe Ser His 325
3301012695DNAHomo sapiens 10cgcgccgggg cctggtgctc ggtcggcggg
tgctgccgct ttaagcgggg gcgggactgc 60gcgcggccga gcggttgcga cgagggctcg
gctgggggtc gccggggtcg cgggccgggc 120ctgcaggagc cgggccgccg aggtcggggc
tggttgaact catggacctg atacttttct 180cttgagaagc aaaccagccc aaaagaaaaa
tggcgtttgt tgcaacacag ggggccacgg 240tggttgacca gaccactttg atgaaaaagt
accttcagtt tgtggcagct ctcacagatg 300tgaatacacc tgatgaaaca aagttgaaaa
tgatgcaaga agttagtgaa aattttgaga 360atgtcacgtc atctcctcag tattctacat
tcctagaaca tatcatccct cgattcctta 420catttctcca agatggagaa gttcagtttc
ttcaggagaa accagcacag caactgcgga 480agctcgtact tgaaataatt catagaatac
caaccaacga acatcttcgt cctcacacaa 540aaaatgtttt gtctgtgatg tttcgctttt
tagagacgga aaatgaagaa aatgttctta 600tttgtctaag aataattatt gagctacaca
aacagttcag gccaccgatc acacaagaaa 660ttcatcattt tctggatttt gtgaaacaga
tttacaagga gcttccaaaa gtagtgaacc 720gctactttga gaaccctcaa gtgatccccg
agaacacagt gcctccccca gaaatggttg 780gtatgataac aacgattgct gtgaaagtca
acccggagcg tgaggacagt gagactcgaa 840cacattccat cattccgagg ggatcacttt
ctctgaaagt gttggcagaa ttgcccatta 900ttgttgtttt aatgtatcag ctctacaaac
tgaacatcca caatgttgtt gctgagtttg 960tgcccttgat catgaacacc attgccattc
aggtgtctgc acaagcgagg caacataagc 1020tttacaacaa ggagttgtat gctgacttca
ttgctgctca gattaaaaca ttgtcatttt 1080tagcttacat tatcaggatt taccaggagt
tggtgactaa gtattctcag cagatggtga 1140aaggaatgct ccagttactt tcaaattgtc
cagcagagac tgcacacctc agaaaggagc 1200ttctgattgc tgccaaacac atcctcacca
cagagctgag aaaccagttc attccttgca 1260tggacaagct gtttgatgaa tccatactaa
ttggctcagg atatactgcc agagagactc 1320taaggcccct cgcctacagc acgctggccg
acctcgtgca ccatgtccgc cagcacctgc 1380ccctcagcga cctctccctc gccgtccagc
tcttcgccaa gaacatcgac gatgagtccc 1440tgcccagcag catccagacc atgtcctgca
agctcctgct gaacctggtg gactgcatcc 1500gttccaagag cgagcaggag agtggcaatg
ggagagacgt cctgatgcgg atgctggagg 1560ttttcgttct caaattccac acaattgctc
ggtaccagct ctctgccatt tttaagaagt 1620gtaagcctca gtcagaactt ggagccgtgg
aagcagctct gcctggggtg cccactgccc 1680ctgcagctcc tggccctgct ccctccccag
cccctgtccc tgccccacct ccacccccgc 1740ccccaccccc acctgccacc cctgtgaccc
cggcccccgt gcctcccttc gagaagcaag 1800gagaaaagga caaggaagac aagcagacat
tccaagtcac agactgtcga agtttggtca 1860aaaccttggt gtgtggtgtc aagacaatca
cgtggggcat aacatcatgc aaagcacctg 1920gtgaagctca gttcattccc aacaagcagt
tacaacccaa agagacacag atttacatca 1980aacttgtgaa atatgcaatg caagctttag
atatttatca ggtccagata gcaggaaatg 2040gacagacata catccgtgtg gccaactgcc
agactgtgag aatgaaagag gagaaggagg 2100tattggagca tttcgctggt gtgttcacaa
tgatgaaccc cttaacgttc aaagaaatct 2160tccaaactac ggtcccttat atggtggaga
gaatctcaaa aaattatgct cttcagattg 2220ttgccaattc cttcttggca aatcctacta
cctctgctct gtttgctacg attctggtgg 2280aatatctcct tgatcgcctg ccagaaatgg
gctccaacgt ggagctctcc aacctgtacc 2340tcaagctgtt caagctggtc tttggctctg
tctccctctt tgcagctgaa aatgaacaaa 2400tgctgaagcc tcacttgcac aagattgtga
acagctctat ggagctcgcg cagactgcca 2460aggaacccta caactacttc ttgctgctac
gggcgctgtt tcgctctatt ggtggaggta 2520gccacgatct cttgtatcag gagttcttgc
ctctccttcc aaacctcctg caagggctga 2580acatgcttca gagtggcctg cacaagcagc
acatgaagga cctctttgtg gagctgtgtc 2640tcaccgtccc tgtgcggctg agctcgcttt
tgccgtacct gcccatgctt atggatccct 2700tggtgtctgc actcaatggg tctcagacat
tggtcagcca aggcctcagg acgctggagc 2760tgtgtgtgga caacctgcag cccgacttcc
tctacgacca catccagccg gtgcgcgcag 2820agctcatgca ggctctgtgg cgcaccttac
gcaaccctgc tgacagcatc tcccacgtgg 2880cctaccgtgt gctcggtaag tttggcggca
gtaacaggaa gatgctgaag gagtcgcaga 2940agctgcacta cgttgtgacc gaggttcagg
gccccagcat cactgtggag ttttccgact 3000gcaaagcttc tctccagctc cccatggaga
aggccattga aactgctctg gactgcctga 3060aaagcgccaa cactgagccc tactaccgga
ggcaggcgtg ggaagtgatc aaatgcttcc 3120tggtggccat gatgagcctg gaggacaaca
agcacgcact ctaccagctc ctggcacacc 3180ccaactttac agaaaagacc atccccaatg
ttatcatctc acatcgctac aaagcccagg 3240acactccagc ccggaagact tttgagcagg
ccctgacagg cgccttcatg tctgctgtca 3300ttaaggacct gcggcccagc gccctgccct
ttgtcgccag cttgatccgc cactatacga 3360tggtggcagt cgcccagcag tgtggccctt
tcttgctgcc ttgctaccag gtgggcagcc 3420agcccagcac agccatgttt cacagtgaag
aaaatggctc gaaaggaatg gatcctttgg 3480ttctcattga tgcaattgct atttgtatgg
catatgaaga aaaggagctt tgcaaaatcg 3540gggaggtggc cctagctgtg atatttgatg
ttgcaagtat catcctgggc tccaaggaga 3600gggcctgcca gctgcccctg ttttcttaca
tcgtggagcg cctgtgtgca tgttgttatg 3660aacaggcgtg gtatgcaaag ctggggggtg
tggtgtctat taagtttctc atggagcggc 3720tgcctctcac ttgggttctc cagaaccagc
agacattcct gaaagcactt ctctttgtca 3780tgatggactt aactggagag gtttccaatg
gggcagtcgc tatggcaaag accacgctgg 3840agcagcttct gatgcggtgc gcaacgcctt
taaaagacga ggagagagcc gaagagatcg 3900tggccgccca ggaaaagtct ttccaccatg
tgacacacga cttggttcga gaagtcacct 3960ctccaaactc cactgtgagg aagcaggcca
tgcattcgct gcaggtgttg gcccaggtca 4020ctgggaagag tgtcacggtg atcatggaac
cccacaaaga ggtcctgcag gatatggtcc 4080cccctaagaa gcacctgctc cgacaccagc
ctgccaacgc acagattggc ctgatggagg 4140ggaacacgtt ctgtaccacg ttgcagccca
ggctcttcac aatggacctt aacgtggtgg 4200agcataaggt gttctacaca gagctgttga
atttgtgtga ggctgaagat tcagctttaa 4260caaagctgcc ctgttataaa agccttccgt
cactcgtacc tttacgaatt gcggcattaa 4320atgcacttgc tgcctgcaat taccttcctc
agtccaggga gaaaatcatc gctgcactct 4380tcaaagccct gaattccacc aatagtgagc
tccaagaggc cggagaagcc tgtatgagaa 4440agtttttaga aggtgctacc atagaagtcg
atcaaatcca cacacatatg cgacctttgc 4500tgatgatgct gggagattac cggagcttga
cgctgaatgt tgtgaatcgc ctgacttcgg 4560tcacgaggct cttcccaaat tccttcaatg
ataaattttg tgatcagatg atgcaacatc 4620tgcgcaagtg gatggaagtg gtggtgatca
cccacaaagg gggccagagg agcgacggaa 4680acgaaagcat ttccgagtgc gggagatgtc
ccttgtctcc attctgtcag tttgaggaaa 4740tgaagatttg ctcagcaatt ataaaccttt
ttcatctgat cccggctgct cctcagacac 4800tggtgaagcc tttgctagag gttgtcatga
aaacggagcg ggcgatgctg atcgaggcgg 4860ggagtccatt ccgagagccc ctgatcaagt
tcctgactcg acatccctcg cagacagtgg 4920agctgttcat gatggaagcc acactgaacg
atccccagtg gagcagaatg tttatgagtt 4980ttttaaaaca caaagacgcc agacctctgc
gggatgtgct ggctgccaac cccaacaggt 5040tcatcaccct gctgctgccg gggggtgccc
agacggctgt gcgccccggt tcgcccagca 5100ccagcaccat gcgcctggac ctccagttcc
aggccatcaa gatcataagc attatagtga 5160aaaacgatga ctcctggctg gccagccagc
actctctggt gagccagttg cgacgtgtgt 5220gggtgagtga gaacttccaa gagaggcacc
gcaaggagaa catggcagcc accaactgga 5280aggagcccaa gctgctggcc tactgcctgc
tgaactactg caaaaggaat tacggagata 5340tagaattgct gttccagctg ctccgagcct
ttactggtcg ttttctctgc aacatgacat 5400tcttaaaaga gtatatggag gaagagattc
ccaaaaatta cagcatcgct cagaaacgtg 5460ccctgttctt tcgctttgta gacttcaacg
accccaactt cggagatgaa ttaaaagcta 5520aagttctgca gcatatcttg aatcctgctt
tcttgtacag ctttgagaag ggggaaggag 5580agcagctctt gggacctccc aatccagaag
gagataaccc agaaagcatc accagtgtgt 5640ttattaccaa ggtcctggac cccgagaagc
aggcggacat gctggactcg ctgcggatct 5700acctgctgca gtacgccacg ctgctggtgg
agcacgcccc ccaccacatc catgacaaca 5760acaagaaccg caacagcaag ctgcgccgcc
tcatgacctt cgcctggccc tgcctgctct 5820ccaaggcctg cgtggaccca gcctgcaagt
acagcggaca cttgctcctg gcgcacatta 5880tcgccaaatt cgccatacac aagaagatcg
tcctgcaggt ttttcatagt ctcctcaagg 5940ctcacgcaat ggaagctcga gcgatcgtca
gacaggcgat ggccattctg accccggcgg 6000tgccggccag gatggaggac gggcaccaga
tgctgaccca ctggacccgg aagatcattg 6060tggaggaggg gcacaccgtc ccgcagctgg
tccacattct gcacctgata gtgcaacact 6120tcaaggtgta ctacccggta cggcaccact
tggtgcagca catggtgagc gccatgcaga 6180ggctgggctt cacgcccagt gtcaccatcg
agcagaggcg gctggccgtg gacctgtctg 6240aagtcgtcat caagtgggag ctgcagagga
tcaaggacca gcagccggat tcagatatgg 6300acccaaattc cagtggagaa ggagtcaatt
ctgtctcatc ctccattaag agaggcctgt 6360ccgtggattc tgcccaggaa gtgaaacgct
ttaggacggc caccggagcc atcagtgcag 6420tctttgggag gagccagtcg ctacctggag
cagactctct cctcgccaag cccattgaca 6480agcagcacac agacactgtg gtgaacttcc
ttatccgcgt ggcctgtcag gttaatgaca 6540acaccaacac agcggggtcc cctggggagg
tgctctctcg ccggtgtgtg aaccttctga 6600agactgcgtt gcggccagac atgtggccca
agtccgaact caagctgcag tggttcgaca 6660agctgctgat gactgtggag cagccaaacc
aagtgaacta tgggaatatc tgcacgggcc 6720tagaagtgct gagcttcctg ctaactgtcc
tccagtcccc agccatcctc agtagcttca 6780aacctctgca gcgtggaatt gccgcctgca
tgacatgtgg aaacaccaag gtgttgcgag 6840ccgtccacag ccttctctcg cgcctgatga
gcattttccc aacagagccg agtacttcca 6900gtgtggcctc caaatatgaa gagctggagt
gcctctacgc agccgtcgga aaggtcatct 6960atgaagggct caccaactac gagaaggcca
ccaatgccaa tccctcccag ctcttcggga 7020cccttatgat cctcaagtct gcctgcagca
acaaccccag ctacatagac aggctgatct 7080ccgtctttat gcgctccctg cagaagatgg
tccgggagca tttaaaccct caggcagcgt 7140caggaagcac cgaagccacc tcaggtacaa
gcgagctggt gatgctgagt ctggagctgg 7200tgaagacgcg cctggcagtg atgagcatgg
agatgcggaa gaacttcatc caggccatcc 7260tgacatccct catcgaaaaa tcaccagatg
ccaaaatcct ccgggctgtg gtcaaaatcg 7320tggaagaatg ggtcaagaat aactccccaa
tggcagccaa tcagacacct acactccggg 7380agaagtccat tttgcttgtg aagatgatga
cttacataga aaaacgcttt ccggaagacc 7440ttgaattaaa tgcccagttt ttagatcttg
ttaactatgt ctacagggat gagaccctct 7500ctggcagcga gctgacggcg aaacttgagc
ctgcctttct ctctgggctg cgctgtgccc 7560agccactcat cagggcaaag tttttcgagg
tttttgacaa ctccatgaaa cgtcgtgtct 7620acgagcgctt gctctatgtg acctgttcgc
agaactggga agccatgggg aaccacttct 7680ggatcaagca gtgcattgag ctgcttctgg
ccgtgtgtga gaagagcacc cccattggca 7740ccagctgcca aggagccatg ctcccgtcca
tcaccaacgt catcaacctg gccgatagcc 7800acgaccgtgc cgccttcgcc atggtcacac
atgtcaagca ggagccccgg gagcgggaga 7860acagcgagtc caaagaggag gatgtagaga
tagacatcga actagctcct ggggatcaga 7920ccagcacgcc caaaaccaaa gaactttcag
aaaaggacat tggaaaccag ctgcacatgc 7980taaccaacag gcacgacaag tttctggaca
ctctccgaga ggtgaagact ggagcgctgc 8040tcagcgcttt cgttcagctg tgccacattt
ccacgacgct ggcagagaag acgtgggtcc 8100agcttttccc cagattgtgg aagatcctct
ctgacagaca gcagcatgca ctcgcgggtg 8160agataagtcc atttctgtgc agcggcagtc
accaggtgca gcgggactgc cagcccagcg 8220cgctgaactg ctttgtggaa gccatgtccc
agtgcgtgcc gccaatcccc atccgaccct 8280gcgtcctgaa gtacctgggg aagacacaca
acctctggtt ccggtccacg ctgatgttgg 8340agcaccaggc ttttgaaaag ggtctgagtc
ttcagattaa gccgaagcaa acaacggagt 8400tttatgagca ggagagcatc accccgccgc
agcaggagat actggattcc cttgcggagc 8460tttactccct gttacaagag gaagatatgt
gggctggtct gtggcagaag cggtgcaagt 8520actcggagac agcgactgcg attgcttacg
agcagcacgg gttctttgag caggcacaag 8580aatcctatga aaaggcaatg gataaagcca
aaaaagaaca tgagaggagt aacgcctccc 8640ctgctatttt ccctgaatac cagctctggg
aagaccactg gattcgatgc tccaaggaat 8700tgaaccagtg ggaagccctg acggagtacg
gtcagtccaa aggccacatc aacccctacc 8760tcgtcctgga gtgcgcctgg cgggtgtcca
actggactgc catgaaggag gcgctggtgc 8820aggtggaagt gagctgtccg aaggagatgg
cctggaaggt gaacatgtac cgcggatacc 8880tggccatctg ccaccccgag gagcagcagc
tcagcttcat cgagcgcctg gtggagatgg 8940ccagcagcct ggccatccgc gagtggcggc
ggctgcccca cgtagtgtcc cacgtgcaca 9000cgcctctcct acaggcagcc cagcaaatca
tcgaactcca ggaagctgca caaatcaacg 9060caggcttaca gccaaccaac ctgggaagga
acaacagcct gcacgacatg aagacggtgg 9120tgaagacctg gaggaaccga ctgcccatcg
tgtctgacga cttgtcccac tggagcagca 9180tcttcatgtg gaggcagcat cattaccagg
gtaaaccgac ctggtccggc atgcattcat 9240catcgattgt aactgcctat gagaatagct
ctcagcatga tcccagttca aataacgcta 9300tgcttggggt tcatgcatca gcttcagcga
tcatccagta tggaaaaatc gcccggaaac 9360aaggactggt caatgtagct ctggatatat
taagtcggat tcatactatt ccaactgttc 9420ctatcgtgga ttgcttccag aagattcgac
agcaagttaa atgctacctc cagctggcag 9480gcgtcatggg caaaaacgag tgcatgcagg
gccttgaagt tattgaatct acaaatttaa 9540aatacttcac aaaagagatg acagccgaat
tttatgcact gaagggaatg ttcttggctc 9600agatcaacaa gtccgaggag gcaaacaaag
ccttctctgc agctgtgcag atgcacgatg 9660tgctggtgaa agcctgggcc atgtggggcg
actacctgga gaacatcttt gtgaaggagc 9720ggcagctgca cctgggcgtg tctgccatca
cctgctacct gcacgcctgc cggcatcaga 9780acgagagcaa atcgaggaaa tacttagcca
aggtgctgtg gcttttgagt tttgatgatg 9840acaaaaacac tttggcagat gccgtcgaca
agtactgcat tggtgtgcca cccatccagt 9900ggctggcctg gatcccacag ctgctcacct
gcctggttgg ctcggaggga aagctgctct 9960tgaacctcat tagccaggtt ggacgcgtgt
atccccaagc ggtctacttt cccatccgga 10020ccctgtacct gaccctgaaa atagaacagc
gggaacgcta caagagcgat ccagggccca 10080taagagcaac agcacccatg tggcgctgca
gccgaatcat gcacatgcag cgagagctcc 10140accccaccct tctgtcttcc ctggaaggca
tcgtcgatca gatggtctgg ttcagagaaa 10200attggcatga agaggttctc aggcagctcc
aacagggcct ggcgaaatgt tactccgtgg 10260cgtttgagaa aagtggagcg gtgtccgatg
ctaaaatcac cccccacact ctcaattttg 10320tgaagaagtt ggtgagcacg tttggggtgg
gcctggagaa tgtgtccaac gtctcgacca 10380tgttctccag cgcagcctct gagtctctgg
cccggcgggc gcaggccact gcacaagacc 10440ctgtctttca gaagctgaaa ggccagttca
cgacggattt tgacttcagc gttccaggat 10500ccatgaagct tcataatctt atttctaagt
tgaaaaagtg gatcaaaatc ttggaggcca 10560agaccaagca actccccaaa ttcttcctca
tagaggaaaa gtgccggttc ttgagcaatt 10620tctcggcaca gacagctgaa gtggaaattc
ctggggagtt tctgatgcca aagccaacgc 10680attattacat caagattgca cggttcatgc
cccgggtaga gattgtgcag aagcacaaca 10740ccgcagcccg gcggctgtac atccggggac
acaatggcaa gatctaccca tacctcgtca 10800tgaacgacgc ctgcctcaca gagtcacggc
gagaggagcg tgtgttgcag ctgctgcgtc 10860tgctgaaccc ctgtttggag aagagaaagg
agaccaccaa gaggcacttg tttttcacag 10920tgccccgggt tgtggcagtt tccccacaga
tgcgcctcgt ggaggacaac ccctcttcac 10980tttcccttgt ggagatctac aagcagcgct
gcgccaagaa gggcatcgag catgacaacc 11040ccatctcccg ttactatgac cggctggcta
cggtgcaggc gcggggaacc caagccagcc 11100accaggtcct ccgcgacatc ctcaaggagg
ttcagagtaa catggtgccg cgcagcatgc 11160tcaaggagtg ggcgctgcac accttcccca
atgccacgga ctactggacg ttccggaaga 11220tgttcaccat ccagctggct ctgataggct
tcgcggaatt cgtcctgcat ttaaatagac 11280tcaaccccga gatgttacag atcgctcagg
acactggcaa actgaatgtt gcctactttc 11340gatttgacat aaacgacgcg actggagacc
tggatgccaa ccgtcctgtc ccatttcgac 11400tcacgcccaa catttctgag tttctgacca
ccatcggggt ctccggcccg ttgacagcgt 11460ccatgattgc ggtcgcccgg tgcttcgccc
agccaaactt taaggtggat ggcattctga 11520aaacggttct ccgggacgag atcattgctt
ggcacaaaaa aacacaagag gacacgtcct 11580ctcctctctc ggccgccggg cagccagaga
acatggacag ccagcaactg gtgtccctgg 11640ttcagaaagc cgtcaccgcc atcatgaccc
gcctgcacaa cctcgcccag ttcgaaggcg 11700gggaaagcaa ggtgaacacc ctggtggccg
cggcaaacag cctggacaat ctgtgccgca 11760tggaccccgc ctggcacccc tggctgtgac
tgtggccgcc acggccaccc ggaatgtgaa 11820gggcgctccg ggctctgagc ccgcagcttt
tacgacttct ccctgcctcg ttccttatat 11880tcacagaagc cccatagttt cactgggttg
cggttatttt cctggtagtt tgcgtgtaag 11940aaagggagaa tatagtttta gaggaagctg
aactatgacg atgctgggcg aagcggttgg 12000aaatggcaga gctgaaactt attccaagct
ttcaaaataa tcttttaaga agccaggatt 12060ctccggtctg gaatttctga gtgagtcctt
tttttatggt gtcctccctc tgtgaatgta 12120caggcggaac tgtacgaaca gctcccttcc
atccattttt aactctttcg gaaataacac 12180ctcacagcag cttcgtgctt ttgtacagac
ctttgtaaca agtgtacaga aaactcattt 12240tgtttgagaa acaggagttg atgaacccat
catgctggtt tttctctgag cacaaagttt 12300taggctgtac acagccagcc ttgggaatct
cgttgagcgt tcggcgtgga tccacggggc 12360caggccaccc tgcgggagcg ccacacgcat
ccacttcgga ttcagtgggt gaagacagaa 12420ctctgagagt ctgcaggcgg ctcctgtgct
ttttatttct ggctcttcgg atgtcttcta 12480gacatttact atcactgcac ctgaagaaaa
aatcactttt accttcctaa tttaaaaaga 12540caaaacagaa atgtacgttc cttcgctagc
tttagtcttt ctgttcccat ttttataaat 12600ctgagcattg ataatgttct atctaaattt
gtacagtgtg attttttttt ttagaataaa 12660tattttataa aagggttaaa aaaaaaaaaa
aaaaa 12695113858PRTHomo sapiens 11Met Ala
Phe Val Ala Thr Gln Gly Ala Thr Val Val Asp Gln Thr Thr1 5
10 15Leu Met Lys Lys Tyr Leu Gln Phe
Val Ala Ala Leu Thr Asp Val Asn 20 25
30Thr Pro Asp Glu Thr Lys Leu Lys Met Met Gln Glu Val Ser Glu
Asn 35 40 45Phe Glu Asn Val Thr
Ser Ser Pro Gln Tyr Ser Thr Phe Leu Glu His 50 55
60Ile Ile Pro Arg Phe Leu Thr Phe Leu Gln Asp Gly Glu Val
Gln Phe65 70 75 80Leu
Gln Glu Lys Pro Ala Gln Gln Leu Arg Lys Leu Val Leu Glu Ile
85 90 95Ile His Arg Ile Pro Thr Asn
Glu His Leu Arg Pro His Thr Lys Asn 100 105
110Val Leu Ser Val Met Phe Arg Phe Leu Glu Thr Glu Asn Glu
Glu Asn 115 120 125Val Leu Ile Cys
Leu Arg Ile Ile Ile Glu Leu His Lys Gln Phe Arg 130
135 140Pro Pro Ile Thr Gln Glu Ile His His Phe Leu Asp
Phe Val Lys Gln145 150 155
160Ile Tyr Lys Glu Leu Pro Lys Val Val Asn Arg Tyr Phe Glu Asn Pro
165 170 175Gln Val Ile Pro Glu
Asn Thr Val Pro Pro Pro Glu Met Val Gly Met 180
185 190Ile Thr Thr Ile Ala Val Lys Val Asn Pro Glu Arg
Glu Asp Ser Glu 195 200 205Thr Arg
Thr His Ser Ile Ile Pro Arg Gly Ser Leu Ser Leu Lys Val 210
215 220Leu Ala Glu Leu Pro Ile Ile Val Val Leu Met
Tyr Gln Leu Tyr Lys225 230 235
240Leu Asn Ile His Asn Val Val Ala Glu Phe Val Pro Leu Ile Met Asn
245 250 255Thr Ile Ala Ile
Gln Val Ser Ala Gln Ala Arg Gln His Lys Leu Tyr 260
265 270Asn Lys Glu Leu Tyr Ala Asp Phe Ile Ala Ala
Gln Ile Lys Thr Leu 275 280 285Ser
Phe Leu Ala Tyr Ile Ile Arg Ile Tyr Gln Glu Leu Val Thr Lys 290
295 300Tyr Ser Gln Gln Met Val Lys Gly Met Leu
Gln Leu Leu Ser Asn Cys305 310 315
320Pro Ala Glu Thr Ala His Leu Arg Lys Glu Leu Leu Ile Ala Ala
Lys 325 330 335His Ile Leu
Thr Thr Glu Leu Arg Asn Gln Phe Ile Pro Cys Met Asp 340
345 350Lys Leu Phe Asp Glu Ser Ile Leu Ile Gly
Ser Gly Tyr Thr Ala Arg 355 360
365Glu Thr Leu Arg Pro Leu Ala Tyr Ser Thr Leu Ala Asp Leu Val His 370
375 380His Val Arg Gln His Leu Pro Leu
Ser Asp Leu Ser Leu Ala Val Gln385 390
395 400Leu Phe Ala Lys Asn Ile Asp Asp Glu Ser Leu Pro
Ser Ser Ile Gln 405 410
415Thr Met Ser Cys Lys Leu Leu Leu Asn Leu Val Asp Cys Ile Arg Ser
420 425 430Lys Ser Glu Gln Glu Ser
Gly Asn Gly Arg Asp Val Leu Met Arg Met 435 440
445Leu Glu Val Phe Val Leu Lys Phe His Thr Ile Ala Arg Tyr
Gln Leu 450 455 460Ser Ala Ile Phe Lys
Lys Cys Lys Pro Gln Ser Glu Leu Gly Ala Val465 470
475 480Glu Ala Ala Leu Pro Gly Val Pro Thr Ala
Pro Ala Ala Pro Gly Pro 485 490
495Ala Pro Ser Pro Ala Pro Val Pro Ala Pro Pro Pro Pro Pro Pro Pro
500 505 510Pro Pro Pro Ala Thr
Pro Val Thr Pro Ala Pro Val Pro Pro Phe Glu 515
520 525Lys Gln Gly Glu Lys Asp Lys Glu Asp Lys Gln Thr
Phe Gln Val Thr 530 535 540Asp Cys Arg
Ser Leu Val Lys Thr Leu Val Cys Gly Val Lys Thr Ile545
550 555 560Thr Trp Gly Ile Thr Ser Cys
Lys Ala Pro Gly Glu Ala Gln Phe Ile 565
570 575Pro Asn Lys Gln Leu Gln Pro Lys Glu Thr Gln Ile
Tyr Ile Lys Leu 580 585 590Val
Lys Tyr Ala Met Gln Ala Leu Asp Ile Tyr Gln Val Gln Ile Ala 595
600 605Gly Asn Gly Gln Thr Tyr Ile Arg Val
Ala Asn Cys Gln Thr Val Arg 610 615
620Met Lys Glu Glu Lys Glu Val Leu Glu His Phe Ala Gly Val Phe Thr625
630 635 640Met Met Asn Pro
Leu Thr Phe Lys Glu Ile Phe Gln Thr Thr Val Pro 645
650 655Tyr Met Val Glu Arg Ile Ser Lys Asn Tyr
Ala Leu Gln Ile Val Ala 660 665
670Asn Ser Phe Leu Ala Asn Pro Thr Thr Ser Ala Leu Phe Ala Thr Ile
675 680 685Leu Val Glu Tyr Leu Leu Asp
Arg Leu Pro Glu Met Gly Ser Asn Val 690 695
700Glu Leu Ser Asn Leu Tyr Leu Lys Leu Phe Lys Leu Val Phe Gly
Ser705 710 715 720Val Ser
Leu Phe Ala Ala Glu Asn Glu Gln Met Leu Lys Pro His Leu
725 730 735His Lys Ile Val Asn Ser Ser
Met Glu Leu Ala Gln Thr Ala Lys Glu 740 745
750Pro Tyr Asn Tyr Phe Leu Leu Leu Arg Ala Leu Phe Arg Ser
Ile Gly 755 760 765Gly Gly Ser His
Asp Leu Leu Tyr Gln Glu Phe Leu Pro Leu Leu Pro 770
775 780Asn Leu Leu Gln Gly Leu Asn Met Leu Gln Ser Gly
Leu His Lys Gln785 790 795
800His Met Lys Asp Leu Phe Val Glu Leu Cys Leu Thr Val Pro Val Arg
805 810 815Leu Ser Ser Leu Leu
Pro Tyr Leu Pro Met Leu Met Asp Pro Leu Val 820
825 830Ser Ala Leu Asn Gly Ser Gln Thr Leu Val Ser Gln
Gly Leu Arg Thr 835 840 845Leu Glu
Leu Cys Val Asp Asn Leu Gln Pro Asp Phe Leu Tyr Asp His 850
855 860Ile Gln Pro Val Arg Ala Glu Leu Met Gln Ala
Leu Trp Arg Thr Leu865 870 875
880Arg Asn Pro Ala Asp Ser Ile Ser His Val Ala Tyr Arg Val Leu Gly
885 890 895Lys Phe Gly Gly
Ser Asn Arg Lys Met Leu Lys Glu Ser Gln Lys Leu 900
905 910His Tyr Val Val Thr Glu Val Gln Gly Pro Ser
Ile Thr Val Glu Phe 915 920 925Ser
Asp Cys Lys Ala Ser Leu Gln Leu Pro Met Glu Lys Ala Ile Glu 930
935 940Thr Ala Leu Asp Cys Leu Lys Ser Ala Asn
Thr Glu Pro Tyr Tyr Arg945 950 955
960Arg Gln Ala Trp Glu Val Ile Lys Cys Phe Leu Val Ala Met Met
Ser 965 970 975Leu Glu Asp
Asn Lys His Ala Leu Tyr Gln Leu Leu Ala His Pro Asn 980
985 990Phe Thr Glu Lys Thr Ile Pro Asn Val Ile
Ile Ser His Arg Tyr Lys 995 1000
1005Ala Gln Asp Thr Pro Ala Arg Lys Thr Phe Glu Gln Ala Leu Thr
1010 1015 1020Gly Ala Phe Met Ser Ala
Val Ile Lys Asp Leu Arg Pro Ser Ala 1025 1030
1035Leu Pro Phe Val Ala Ser Leu Ile Arg His Tyr Thr Met Val
Ala 1040 1045 1050Val Ala Gln Gln Cys
Gly Pro Phe Leu Leu Pro Cys Tyr Gln Val 1055 1060
1065Gly Ser Gln Pro Ser Thr Ala Met Phe His Ser Glu Glu
Asn Gly 1070 1075 1080Ser Lys Gly Met
Asp Pro Leu Val Leu Ile Asp Ala Ile Ala Ile 1085
1090 1095Cys Met Ala Tyr Glu Glu Lys Glu Leu Cys Lys
Ile Gly Glu Val 1100 1105 1110Ala Leu
Ala Val Ile Phe Asp Val Ala Ser Ile Ile Leu Gly Ser 1115
1120 1125Lys Glu Arg Ala Cys Gln Leu Pro Leu Phe
Ser Tyr Ile Val Glu 1130 1135 1140Arg
Leu Cys Ala Cys Cys Tyr Glu Gln Ala Trp Tyr Ala Lys Leu 1145
1150 1155Gly Gly Val Val Ser Ile Lys Phe Leu
Met Glu Arg Leu Pro Leu 1160 1165
1170Thr Trp Val Leu Gln Asn Gln Gln Thr Phe Leu Lys Ala Leu Leu
1175 1180 1185Phe Val Met Met Asp Leu
Thr Gly Glu Val Ser Asn Gly Ala Val 1190 1195
1200Ala Met Ala Lys Thr Thr Leu Glu Gln Leu Leu Met Arg Cys
Ala 1205 1210 1215Thr Pro Leu Lys Asp
Glu Glu Arg Ala Glu Glu Ile Val Ala Ala 1220 1225
1230Gln Glu Lys Ser Phe His His Val Thr His Asp Leu Val
Arg Glu 1235 1240 1245Val Thr Ser Pro
Asn Ser Thr Val Arg Lys Gln Ala Met His Ser 1250
1255 1260Leu Gln Val Leu Ala Gln Val Thr Gly Lys Ser
Val Thr Val Ile 1265 1270 1275Met Glu
Pro His Lys Glu Val Leu Gln Asp Met Val Pro Pro Lys 1280
1285 1290Lys His Leu Leu Arg His Gln Pro Ala Asn
Ala Gln Ile Gly Leu 1295 1300 1305Met
Glu Gly Asn Thr Phe Cys Thr Thr Leu Gln Pro Arg Leu Phe 1310
1315 1320Thr Met Asp Leu Asn Val Val Glu His
Lys Val Phe Tyr Thr Glu 1325 1330
1335Leu Leu Asn Leu Cys Glu Ala Glu Asp Ser Ala Leu Thr Lys Leu
1340 1345 1350Pro Cys Tyr Lys Ser Leu
Pro Ser Leu Val Pro Leu Arg Ile Ala 1355 1360
1365Ala Leu Asn Ala Leu Ala Ala Cys Asn Tyr Leu Pro Gln Ser
Arg 1370 1375 1380Glu Lys Ile Ile Ala
Ala Leu Phe Lys Ala Leu Asn Ser Thr Asn 1385 1390
1395Ser Glu Leu Gln Glu Ala Gly Glu Ala Cys Met Arg Lys
Phe Leu 1400 1405 1410Glu Gly Ala Thr
Ile Glu Val Asp Gln Ile His Thr His Met Arg 1415
1420 1425Pro Leu Leu Met Met Leu Gly Asp Tyr Arg Ser
Leu Thr Leu Asn 1430 1435 1440Val Val
Asn Arg Leu Thr Ser Val Thr Arg Leu Phe Pro Asn Ser 1445
1450 1455Phe Asn Asp Lys Phe Cys Asp Gln Met Met
Gln His Leu Arg Lys 1460 1465 1470Trp
Met Glu Val Val Val Ile Thr His Lys Gly Gly Gln Arg Ser 1475
1480 1485Asp Gly Asn Glu Ser Ile Ser Glu Cys
Gly Arg Cys Pro Leu Ser 1490 1495
1500Pro Phe Cys Gln Phe Glu Glu Met Lys Ile Cys Ser Ala Ile Ile
1505 1510 1515Asn Leu Phe His Leu Ile
Pro Ala Ala Pro Gln Thr Leu Val Lys 1520 1525
1530Pro Leu Leu Glu Val Val Met Lys Thr Glu Arg Ala Met Leu
Ile 1535 1540 1545Glu Ala Gly Ser Pro
Phe Arg Glu Pro Leu Ile Lys Phe Leu Thr 1550 1555
1560Arg His Pro Ser Gln Thr Val Glu Leu Phe Met Met Glu
Ala Thr 1565 1570 1575Leu Asn Asp Pro
Gln Trp Ser Arg Met Phe Met Ser Phe Leu Lys 1580
1585 1590His Lys Asp Ala Arg Pro Leu Arg Asp Val Leu
Ala Ala Asn Pro 1595 1600 1605Asn Arg
Phe Ile Thr Leu Leu Leu Pro Gly Gly Ala Gln Thr Ala 1610
1615 1620Val Arg Pro Gly Ser Pro Ser Thr Ser Thr
Met Arg Leu Asp Leu 1625 1630 1635Gln
Phe Gln Ala Ile Lys Ile Ile Ser Ile Ile Val Lys Asn Asp 1640
1645 1650Asp Ser Trp Leu Ala Ser Gln His Ser
Leu Val Ser Gln Leu Arg 1655 1660
1665Arg Val Trp Val Ser Glu Asn Phe Gln Glu Arg His Arg Lys Glu
1670 1675 1680Asn Met Ala Ala Thr Asn
Trp Lys Glu Pro Lys Leu Leu Ala Tyr 1685 1690
1695Cys Leu Leu Asn Tyr Cys Lys Arg Asn Tyr Gly Asp Ile Glu
Leu 1700 1705 1710Leu Phe Gln Leu Leu
Arg Ala Phe Thr Gly Arg Phe Leu Cys Asn 1715 1720
1725Met Thr Phe Leu Lys Glu Tyr Met Glu Glu Glu Ile Pro
Lys Asn 1730 1735 1740Tyr Ser Ile Ala
Gln Lys Arg Ala Leu Phe Phe Arg Phe Val Asp 1745
1750 1755Phe Asn Asp Pro Asn Phe Gly Asp Glu Leu Lys
Ala Lys Val Leu 1760 1765 1770Gln His
Ile Leu Asn Pro Ala Phe Leu Tyr Ser Phe Glu Lys Gly 1775
1780 1785Glu Gly Glu Gln Leu Leu Gly Pro Pro Asn
Pro Glu Gly Asp Asn 1790 1795 1800Pro
Glu Ser Ile Thr Ser Val Phe Ile Thr Lys Val Leu Asp Pro 1805
1810 1815Glu Lys Gln Ala Asp Met Leu Asp Ser
Leu Arg Ile Tyr Leu Leu 1820 1825
1830Gln Tyr Ala Thr Leu Leu Val Glu His Ala Pro His His Ile His
1835 1840 1845Asp Asn Asn Lys Asn Arg
Asn Ser Lys Leu Arg Arg Leu Met Thr 1850 1855
1860Phe Ala Trp Pro Cys Leu Leu Ser Lys Ala Cys Val Asp Pro
Ala 1865 1870 1875Cys Lys Tyr Ser Gly
His Leu Leu Leu Ala His Ile Ile Ala Lys 1880 1885
1890Phe Ala Ile His Lys Lys Ile Val Leu Gln Val Phe His
Ser Leu 1895 1900 1905Leu Lys Ala His
Ala Met Glu Ala Arg Ala Ile Val Arg Gln Ala 1910
1915 1920Met Ala Ile Leu Thr Pro Ala Val Pro Ala Arg
Met Glu Asp Gly 1925 1930 1935His Gln
Met Leu Thr His Trp Thr Arg Lys Ile Ile Val Glu Glu 1940
1945 1950Gly His Thr Val Pro Gln Leu Val His Ile
Leu His Leu Ile Val 1955 1960 1965Gln
His Phe Lys Val Tyr Tyr Pro Val Arg His His Leu Val Gln 1970
1975 1980His Met Val Ser Ala Met Gln Arg Leu
Gly Phe Thr Pro Ser Val 1985 1990
1995Thr Ile Glu Gln Arg Arg Leu Ala Val Asp Leu Ser Glu Val Val
2000 2005 2010Ile Lys Trp Glu Leu Gln
Arg Ile Lys Asp Gln Gln Pro Asp Ser 2015 2020
2025Asp Met Asp Pro Asn Ser Ser Gly Glu Gly Val Asn Ser Val
Ser 2030 2035 2040Ser Ser Ile Lys Arg
Gly Leu Ser Val Asp Ser Ala Gln Glu Val 2045 2050
2055Lys Arg Phe Arg Thr Ala Thr Gly Ala Ile Ser Ala Val
Phe Gly 2060 2065 2070Arg Ser Gln Ser
Leu Pro Gly Ala Asp Ser Leu Leu Ala Lys Pro 2075
2080 2085Ile Asp Lys Gln His Thr Asp Thr Val Val Asn
Phe Leu Ile Arg 2090 2095 2100Val Ala
Cys Gln Val Asn Asp Asn Thr Asn Thr Ala Gly Ser Pro 2105
2110 2115Gly Glu Val Leu Ser Arg Arg Cys Val Asn
Leu Leu Lys Thr Ala 2120 2125 2130Leu
Arg Pro Asp Met Trp Pro Lys Ser Glu Leu Lys Leu Gln Trp 2135
2140 2145Phe Asp Lys Leu Leu Met Thr Val Glu
Gln Pro Asn Gln Val Asn 2150 2155
2160Tyr Gly Asn Ile Cys Thr Gly Leu Glu Val Leu Ser Phe Leu Leu
2165 2170 2175Thr Val Leu Gln Ser Pro
Ala Ile Leu Ser Ser Phe Lys Pro Leu 2180 2185
2190Gln Arg Gly Ile Ala Ala Cys Met Thr Cys Gly Asn Thr Lys
Val 2195 2200 2205Leu Arg Ala Val His
Ser Leu Leu Ser Arg Leu Met Ser Ile Phe 2210 2215
2220Pro Thr Glu Pro Ser Thr Ser Ser Val Ala Ser Lys Tyr
Glu Glu 2225 2230 2235Leu Glu Cys Leu
Tyr Ala Ala Val Gly Lys Val Ile Tyr Glu Gly 2240
2245 2250Leu Thr Asn Tyr Glu Lys Ala Thr Asn Ala Asn
Pro Ser Gln Leu 2255 2260 2265Phe Gly
Thr Leu Met Ile Leu Lys Ser Ala Cys Ser Asn Asn Pro 2270
2275 2280Ser Tyr Ile Asp Arg Leu Ile Ser Val Phe
Met Arg Ser Leu Gln 2285 2290 2295Lys
Met Val Arg Glu His Leu Asn Pro Gln Ala Ala Ser Gly Ser 2300
2305 2310Thr Glu Ala Thr Ser Gly Thr Ser Glu
Leu Val Met Leu Ser Leu 2315 2320
2325Glu Leu Val Lys Thr Arg Leu Ala Val Met Ser Met Glu Met Arg
2330 2335 2340Lys Asn Phe Ile Gln Ala
Ile Leu Thr Ser Leu Ile Glu Lys Ser 2345 2350
2355Pro Asp Ala Lys Ile Leu Arg Ala Val Val Lys Ile Val Glu
Glu 2360 2365 2370Trp Val Lys Asn Asn
Ser Pro Met Ala Ala Asn Gln Thr Pro Thr 2375 2380
2385Leu Arg Glu Lys Ser Ile Leu Leu Val Lys Met Met Thr
Tyr Ile 2390 2395 2400Glu Lys Arg Phe
Pro Glu Asp Leu Glu Leu Asn Ala Gln Phe Leu 2405
2410 2415Asp Leu Val Asn Tyr Val Tyr Arg Asp Glu Thr
Leu Ser Gly Ser 2420 2425 2430Glu Leu
Thr Ala Lys Leu Glu Pro Ala Phe Leu Ser Gly Leu Arg 2435
2440 2445Cys Ala Gln Pro Leu Ile Arg Ala Lys Phe
Phe Glu Val Phe Asp 2450 2455 2460Asn
Ser Met Lys Arg Arg Val Tyr Glu Arg Leu Leu Tyr Val Thr 2465
2470 2475Cys Ser Gln Asn Trp Glu Ala Met Gly
Asn His Phe Trp Ile Lys 2480 2485
2490Gln Cys Ile Glu Leu Leu Leu Ala Val Cys Glu Lys Ser Thr Pro
2495 2500 2505Ile Gly Thr Ser Cys Gln
Gly Ala Met Leu Pro Ser Ile Thr Asn 2510 2515
2520Val Ile Asn Leu Ala Asp Ser His Asp Arg Ala Ala Phe Ala
Met 2525 2530 2535Val Thr His Val Lys
Gln Glu Pro Arg Glu Arg Glu Asn Ser Glu 2540 2545
2550Ser Lys Glu Glu Asp Val Glu Ile Asp Ile Glu Leu Ala
Pro Gly 2555 2560 2565Asp Gln Thr Ser
Thr Pro Lys Thr Lys Glu Leu Ser Glu Lys Asp 2570
2575 2580Ile Gly Asn Gln Leu His Met Leu Thr Asn Arg
His Asp Lys Phe 2585 2590 2595Leu Asp
Thr Leu Arg Glu Val Lys Thr Gly Ala Leu Leu Ser Ala 2600
2605 2610Phe Val Gln Leu Cys His Ile Ser Thr Thr
Leu Ala Glu Lys Thr 2615 2620 2625Trp
Val Gln Leu Phe Pro Arg Leu Trp Lys Ile Leu Ser Asp Arg 2630
2635 2640Gln Gln His Ala Leu Ala Gly Glu Ile
Ser Pro Phe Leu Cys Ser 2645 2650
2655Gly Ser His Gln Val Gln Arg Asp Cys Gln Pro Ser Ala Leu Asn
2660 2665 2670Cys Phe Val Glu Ala Met
Ser Gln Cys Val Pro Pro Ile Pro Ile 2675 2680
2685Arg Pro Cys Val Leu Lys Tyr Leu Gly Lys Thr His Asn Leu
Trp 2690 2695 2700Phe Arg Ser Thr Leu
Met Leu Glu His Gln Ala Phe Glu Lys Gly 2705 2710
2715Leu Ser Leu Gln Ile Lys Pro Lys Gln Thr Thr Glu Phe
Tyr Glu 2720 2725 2730Gln Glu Ser Ile
Thr Pro Pro Gln Gln Glu Ile Leu Asp Ser Leu 2735
2740 2745Ala Glu Leu Tyr Ser Leu Leu Gln Glu Glu Asp
Met Trp Ala Gly 2750 2755 2760Leu Trp
Gln Lys Arg Cys Lys Tyr Ser Glu Thr Ala Thr Ala Ile 2765
2770 2775Ala Tyr Glu Gln His Gly Phe Phe Glu Gln
Ala Gln Glu Ser Tyr 2780 2785 2790Glu
Lys Ala Met Asp Lys Ala Lys Lys Glu His Glu Arg Ser Asn 2795
2800 2805Ala Ser Pro Ala Ile Phe Pro Glu Tyr
Gln Leu Trp Glu Asp His 2810 2815
2820Trp Ile Arg Cys Ser Lys Glu Leu Asn Gln Trp Glu Ala Leu Thr
2825 2830 2835Glu Tyr Gly Gln Ser Lys
Gly His Ile Asn Pro Tyr Leu Val Leu 2840 2845
2850Glu Cys Ala Trp Arg Val Ser Asn Trp Thr Ala Met Lys Glu
Ala 2855 2860 2865Leu Val Gln Val Glu
Val Ser Cys Pro Lys Glu Met Ala Trp Lys 2870 2875
2880Val Asn Met Tyr Arg Gly Tyr Leu Ala Ile Cys His Pro
Glu Glu 2885 2890 2895Gln Gln Leu Ser
Phe Ile Glu Arg Leu Val Glu Met Ala Ser Ser 2900
2905 2910Leu Ala Ile Arg Glu Trp Arg Arg Leu Pro His
Val Val Ser His 2915 2920 2925Val His
Thr Pro Leu Leu Gln Ala Ala Gln Gln Ile Ile Glu Leu 2930
2935 2940Gln Glu Ala Ala Gln Ile Asn Ala Gly Leu
Gln Pro Thr Asn Leu 2945 2950 2955Gly
Arg Asn Asn Ser Leu His Asp Met Lys Thr Val Val Lys Thr 2960
2965 2970Trp Arg Asn Arg Leu Pro Ile Val Ser
Asp Asp Leu Ser His Trp 2975 2980
2985Ser Ser Ile Phe Met Trp Arg Gln His His Tyr Gln Gly Lys Pro
2990 2995 3000Thr Trp Ser Gly Met His
Ser Ser Ser Ile Val Thr Ala Tyr Glu 3005 3010
3015Asn Ser Ser Gln His Asp Pro Ser Ser Asn Asn Ala Met Leu
Gly 3020 3025 3030Val His Ala Ser Ala
Ser Ala Ile Ile Gln Tyr Gly Lys Ile Ala 3035 3040
3045Arg Lys Gln Gly Leu Val Asn Ala Leu Asp Ile Leu Ser
Arg Ile 3050 3055 3060His Thr Ile Pro
Thr Val Pro Ile Val Asp Cys Phe Gln Lys Ile 3065
3070 3075Arg Gln Gln Val Lys Cys Tyr Leu Gln Leu Ala
Gly Val Met Gly 3080 3085 3090Lys Asn
Glu Cys Met Gln Gly Leu Glu Val Ile Glu Ser Thr Asn 3095
3100 3105Leu Lys Tyr Phe Thr Lys Glu Met Thr Ala
Glu Phe Tyr Ala Leu 3110 3115 3120Lys
Gly Met Phe Leu Ala Gln Ile Asn Lys Ser Glu Glu Ala Asn 3125
3130 3135Lys Ala Phe Ser Ala Ala Val Gln Met
His Asp Val Leu Val Lys 3140 3145
3150Ala Trp Ala Met Trp Gly Asp Tyr Leu Glu Asn Ile Phe Val Lys
3155 3160 3165Glu Arg Gln Leu His Leu
Gly Val Ser Ala Ile Thr Cys Tyr Leu 3170 3175
3180His Ala Cys Arg His Gln Asn Glu Ser Lys Ser Arg Lys Tyr
Leu 3185 3190 3195Ala Lys Val Leu Trp
Leu Leu Ser Phe Asp Asp Asp Lys Asn Thr 3200 3205
3210Leu Ala Asp Ala Val Asp Lys Tyr Cys Ile Gly Val Pro
Pro Ile 3215 3220 3225Gln Trp Leu Ala
Trp Ile Pro Gln Leu Leu Thr Cys Leu Val Gly 3230
3235 3240Ser Glu Gly Lys Leu Leu Leu Asn Leu Ile Ser
Gln Val Gly Arg 3245 3250 3255Val Tyr
Pro Gln Ala Val Tyr Phe Pro Ile Arg Thr Leu Tyr Leu 3260
3265 3270Thr Leu Lys Ile Glu Gln Arg Glu Arg Tyr
Lys Ser Asp Pro Gly 3275 3280 3285Pro
Ile Arg Ala Thr Ala Pro Met Trp Arg Cys Ser Arg Ile Met 3290
3295 3300His Met Gln Arg Glu Leu His Pro Thr
Leu Leu Ser Ser Leu Glu 3305 3310
3315Gly Ile Val Asp Gln Met Val Trp Phe Arg Glu Asn Trp His Glu
3320 3325 3330Glu Val Leu Arg Gln Leu
Gln Gln Gly Leu Ala Lys Cys Tyr Ser 3335 3340
3345Val Ala Phe Glu Lys Ser Gly Ala Val Ser Asp Ala Lys Ile
Thr 3350 3355 3360Pro His Thr Leu Asn
Phe Val Lys Lys Leu Val Ser Thr Phe Gly 3365 3370
3375Val Gly Leu Glu Asn Val Ser Asn Val Ser Thr Met Phe
Ser Ser 3380 3385 3390Ala Ala Ser Glu
Ser Leu Ala Arg Arg Ala Gln Ala Thr Ala Gln 3395
3400 3405Asp Pro Val Phe Gln Lys Leu Lys Gly Gln Phe
Thr Thr Asp Phe 3410 3415 3420Asp Phe
Ser Val Pro Gly Ser Met Lys Leu His Asn Leu Ile Ser 3425
3430 3435Lys Leu Lys Lys Trp Ile Lys Ile Leu Glu
Ala Lys Thr Lys Gln 3440 3445 3450Leu
Pro Lys Phe Phe Leu Ile Glu Glu Lys Cys Arg Phe Leu Ser 3455
3460 3465Asn Phe Ser Ala Gln Thr Ala Glu Val
Glu Ile Pro Gly Glu Phe 3470 3475
3480Leu Met Pro Lys Pro Thr His Tyr Tyr Ile Lys Ile Ala Arg Phe
3485 3490 3495Met Pro Arg Val Glu Ile
Val Gln Lys His Asn Thr Ala Ala Arg 3500 3505
3510Arg Leu Tyr Ile Arg Gly His Asn Gly Lys Ile Tyr Pro Tyr
Leu 3515 3520 3525Val Met Asn Asp Ala
Cys Leu Thr Glu Ser Arg Arg Glu Glu Arg 3530 3535
3540Val Leu Gln Leu Leu Arg Leu Leu Asn Pro Cys Leu Glu
Lys Arg 3545 3550 3555Lys Glu Thr Thr
Lys Arg His Leu Phe Phe Thr Val Pro Arg Val 3560
3565 3570Val Ala Val Ser Pro Gln Met Arg Leu Val Glu
Asp Asn Pro Ser 3575 3580 3585Ser Leu
Ser Leu Val Glu Ile Tyr Lys Gln Arg Cys Ala Lys Lys 3590
3595 3600Gly Ile Glu His Asp Asn Pro Ile Ser Arg
Tyr Tyr Asp Arg Leu 3605 3610 3615Ala
Thr Val Gln Ala Arg Gly Thr Gln Ala Ser His Gln Val Leu 3620
3625 3630Arg Asp Ile Leu Lys Glu Val Gln Ser
Asn Met Val Pro Arg Ser 3635 3640
3645Met Leu Lys Glu Trp Ala Leu His Thr Phe Pro Asn Ala Thr Asp
3650 3655 3660Tyr Trp Thr Phe Arg Lys
Met Phe Thr Ile Gln Leu Ala Leu Ile 3665 3670
3675Gly Phe Ala Glu Phe Val Leu His Leu Asn Arg Leu Asn Pro
Glu 3680 3685 3690Met Leu Gln Ile Ala
Gln Asp Thr Gly Lys Leu Asn Val Ala Tyr 3695 3700
3705Phe Arg Phe Asp Ile Asn Asp Ala Thr Gly Asp Leu Asp
Ala Asn 3710 3715 3720Arg Pro Val Pro
Phe Arg Leu Thr Pro Asn Ile Ser Glu Phe Leu 3725
3730 3735Thr Thr Ile Gly Val Ser Gly Pro Leu Thr Ala
Ser Met Ile Ala 3740 3745 3750Val Ala
Arg Cys Phe Ala Gln Pro Asn Phe Lys Val Asp Gly Ile 3755
3760 3765Leu Lys Thr Val Leu Arg Asp Glu Ile Ile
Ala Trp His Lys Lys 3770 3775 3780Thr
Gln Glu Asp Thr Ser Ser Pro Leu Ser Ala Ala Gly Gln Pro 3785
3790 3795Glu Asn Met Asp Ser Gln Gln Leu Val
Ser Leu Val Gln Lys Ala 3800 3805
3810Val Thr Ala Ile Met Thr Arg Leu His Asn Leu Ala Gln Phe Glu
3815 3820 3825Gly Gly Glu Ser Lys Val
Asn Thr Leu Val Ala Ala Ala Asn Ser 3830 3835
3840Leu Asp Asn Leu Cys Arg Met Asp Pro Ala Trp His Pro Trp
Leu 3845 3850 3855
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